Hemoglobin A1c determination method, enzyme to be used therefor, and production method thereof

ABSTRACT

There is provided a method for specifically determining a glycated β-chain N-terminal of glycated hemoglobin using enzymes without a separation operation, and a determination reagent kit therefor. A protease that cleaves a glycated amino acid and/or a glycated peptide from a glycated β-chain N-terminal without substantially cleaving a glycated amino acid or a glycated peptide from a glycated α-chain N-terminal of glycated hemoglobin or a fragment thereof is screened. The method of specifically determining a glycated β-chain N-terminal of glycated hemoglobin and the determination reagent kit are provided by using the protease obtained by the screening method. According to the present invention, a glycated β-chain N-terminal of glycated hemoglobin can specifically be determined without a separation operation.

TECHNICAL FIELD

The present invention relates to a method of specifically determining aglycated β-chain N-terminal of glycated hemoglobin using enzymes withouta separation operation, a determination reagent kit, a protease that maybe used for the specific determination, a production method thereof, ascreening method thereof, and a ketoamine oxidase that may be used forthe specific determination.

BACKGROUND ART

Determining glycated proteins is very important in diagnosing andcontrolling diabetes. In particular, recent studies on hemoglobin A1chave proved that risk of occurrence and progression of complications issignificantly lowered by controlling the level to 7% or less, so thatthe level is often used as an index that is essential in clinicalfields. As a quantification method for hemoglobin A1c, there aregenerally known electrophoresis, ion-exchange chromatography, affinitychromatography, immunization, and enzymatic methods. However, theelectrophoresis and chromatography methods require expensive dedicateddevices, and processing speeds thereof are low, so that those methodsare inappropriate for clinical examinations to process many samples.Meanwhile, the analysis method of the immunization method is relativelyeasy and can be performed in a small amount of time, so that the methodhas rapidly spread in recent years. However, the method is performedusing an antigen-antibody reaction, so that it is problematic that theaccuracy is not always good due to reproducibility and effects ofcoexisting substances.

Meanwhile, the enzymatic method has been suggested as a determinationmethod that requires no dedicated device, has a high processing speed,and is highly accurate, easy, and inexpensive (JP-A-08-336386, WO97/13872, JP-A-2001-95598, JP-A-2000-300294, and Clinical Chemistry49(2): 269-274 (2003)).

Hemoglobin is glycated at an ε-amino group of intramolecular lysine andat α-amino groups of valine in α- and β-chain N-terminals, buthemoglobin A1c is hemoglobin where an α-amino group of valine in ahemoglobin β-chain N-terminal has been glycated (definition accepted asthe international standard in Clinical Chemistry and Laboratory Medicine40(1): 78-89 (2002)). Therefore, in order to specifically determine aglycated β-chain N-terminal of glycated hemoglobin without a separationoperation using a protease and a ketoamine oxidase, it is believed thatspecificity is required in enzymatic reactions of either or both of aprotease and a ketoamine oxidase.

Specifically, glycated hemoglobin has three glycated sites, that is,intramolecular lysine, α-chain N-terminal, and β-chain N-terminal, sothat in order to determine only the glycated β-chain N-terminal, it isnecessary that those enzymes be combined according to specificity asbelow.

That is, in the case where proteases and ketoamine oxidases areclassified into (P1) to (P4) and (K1) to (K5), respectively, it isnecessary that those enzymes be combined as below: <(P1) and (K1) or(K2) or (K3) or (K4)>, <(P2) and (K1) or (K3)>, <(P3) and (K1) or (K2)>,<(P4) and (K1)>, <(P3) and (K5) and (K3)>, and <(P3) and (K5) and (K4)>.

The properties of the respective classified enzymes are as follows.

(P1) cleaves a glycated amino acid and/or a glycated peptide only from aglycated β-chain N-terminal of glycated hemoglobin, (P2) cleaves aglycated amino acid and/or a glycated peptide only from glycated α- andβ-chain N-terminals of glycated hemoglobin, (P3) cleaves a glycatedamino acid and/or a glycated peptide only from a glycated β-chainN-terminal of glycated hemoglobin and a site including a intramolecularlysine, (P4) cleaves a glycated amino acid and/or a glycated peptidefrom a glycated α- and β-chain N-terminals of glycated hemoglobin and asite including a intramolecular lysine, (K1) reacts only with a glycatedamino acid and/or a glycated peptide derived from a glycated β-chainN-terminal of glycated amino acids and/or glycated peptides cleaved fromglycated hemoglobin by a protease to be used in combination, (K2) reactsonly with a glycated amino acid and/or a glycated peptide derived fromglycated α- and β-chain N-terminals of glycated amino acids and/orglycated peptides cleaved from glycated hemoglobin by a protease to beused in combination, (K3) reacts only with a glycated amino acid and/ora glycated peptide derived from a glycated β-chain N-terminal and from asite including intramolecular lysine of glycated amino acids and/orglycated peptides cleaved from glycated hemoglobin by a protease to beused in combination, (K4) reacts with a glycated amino acid and/or aglycated peptide derived from glycated α- and β-chain N-terminals andfrom a site including intramolecular lysine that have been cleaved fromglycated hemoglobin by a protease to be used in combination, and (K5)reacts with a glycated amino acid and/or a glycated peptide from a siteincluding intramolecular lysine without reacting with a glycated aminoacid and/or a glycated peptide derived from a glycated β-chainN-terminal of glycated amino acids and/or glycated peptides cleaved fromglycated hemoglobin by a protease to be used in combination.

However, as proteases for generating a glycated amino acid and/or aglycated peptide, which serves as a substrate for a ketoamine oxidase,from glycated hemoglobin or a fragment thereof, there have already beenknown proteases described in JP-A-08-336386, WO 97/13872,JP-A-2001-95598, JP-A-2001-57897, WO 00/50579, WO 00/61732, ClinicalChemistry 49(2): 269-274 (2003), etc. However, there is no descriptionabout specificity to cleave a glycated amino acid and/or a glycatedpeptide from a glycated β-chain N-terminal without substantiallycleaving a glycated amino acid or a glycated peptide from a glycatedα-chain N-terminal from glycated hemoglobin or a fragment thereof.

Meanwhile, an angiotensin-converting enzyme described inJP-A-2000-300294 is also estimated to react with a β-chain N-terminalglycated tripeptide from its known substrate specificity, but there isno specific description that shows cleavage of a glycated amino acidand/or a glycated peptide from a glycated β-chain N-terminal withoutsubstantially cleaving a glycated amino acid and/or a glycated peptidefrom a glycated α-chain N-terminal of glycated hemoglobin or a fragmentthereof, for example, there is no description that shows results ofquantification of specificity of the angiotensin-converting enzyme to anα-chain N-terminal glycated tripeptide or to a β-chain N-terminalglycated tripeptide.

Meanwhile, in JP-A-2000-300294, there is no description showing thattrypsin, proline-specific endoprotease, and carboxypeptidase P, whichwere used in generating a β-chain N-terminal glycated tripeptide fromglycated hemoglobin, generate no glycated amino acid or no glycatedpeptide derived from a site including intramolecular lysine and aglycated α-chain N-terminal.

Furthermore, the inventors of the present invention have confirmed thatan angiotensin-converting enzyme hardly cleaves fructosyl valine from aβ-chain N-terminal glycated tripeptide under a general reactioncondition, so that the angiotensin-converting enzyme is not consideredto be a protease that cleaves a glycated amino acid and/or a glycatedpeptide from a glycated β-chain N-terminal without substantiallycleaving a glycated amino acid or a glycated peptide from an α-chainN-terminal.

As described above, there has not been known a protease and a reactioncondition for a protease that cleave a glycated amino acid and/or aglycated peptide from a glycated β-chain N-terminal withoutsubstantially cleaving a glycated amino acid or a glycated peptide froma glycated α-chain N-terminal of glycated hemoglobin or a fragmentthereof, that is, a protease that has the above-described specificity(P1) or (P3) or a reaction condition for a protease that is designed soas to accomplish the above-described specificity (P1) or (P3).

Meanwhile, in general, the following screening method is viewed as amethod of screening a protease that cleaves a glycated amino acid and/ora glycated peptide from a glycated β-chain N-terminal withoutsubstantially cleaving a glycated amino acid or a glycated peptide froma glycated α-chain N-terminal of glycated hemoglobin or a fragmentthereof, that is, a screening method for a protease that has theabove-described specificity (P1) or (P3). That is, glycated hemoglobinsare divided into hemoglobin where an α-chain N-terminal has beenglycated and hemoglobin where a β-chain N-terminal has been glycated,and a protease that selectively cleaves a glycated amino acid and/or aglycated peptide only from the hemoglobin where a β-chain N-terminal hasbeen glycated when using those hemoglobins as substrates is searchedusing an enzyme (such as a ketoamine oxidase) that reacts with theglycated amino acid and/or the glycated peptide cleaved by the protease,based on coloring. However, the glycation rate of a glycated hemoglobinproduct existing in nature is low (about 5%). Therefore, the yield inseparating hemoglobin where an α-chain N-terminal has been glycated andhemoglobin where a β-chain N-terminal has been glycated was extremelylow, and it was difficult to detect the activity of the protease usingthose substances as substrates because hemoglobin is red. As describedabove, an easy and effective screening method has never been known.

On the other hand, ketoamine oxidases include the following enzymes.

1) A ketoamine oxidase that is derived from a microorganism belonging tothe genus Fusarium (JP-A-07-289253), the genus Gibberella, the genusCandida (JP-A-06-46846), or the genus Aspergillus (WO 97/20039) andmainly reacts with ε-1-deoxyfructosyl-L-lysine (hereinafter alsoreferred to as FK) or a peptide including it and fructosyl valine(hereinafter also referred to as FV),

2) a ketoamine oxidase that is derived from a microorganism belonging tothe genus Corynebacterium (JP-A-61-280297), the genus Penicillium(JP-A-08-336386), or the genus Trichosporon (JP-A-2000-245454) andmainly reacts with FV. In general, a step for cleaving FV from ahemoglobin β-chain N-terminal glycated peptide using a protease hasdisadvantages in that the reaction hardly proceeds in general and mustbe performed using a large amount of enzymes for a long time, so that,in order to overcome such disadvantages, a ketoamine oxidase that reactsalso with a glycated peptide cleaved from a hemoglobin β-chainN-terminal glycated peptide by a protease has been required as aketoamine oxidase to be used in determining hemoglobin A1c. Thus,

3) a mutant ketoamine oxidase derived from CorynebacteriumJP-A-2001-95598) and a ketoamine oxidase that is derived from amicroorganism belonging to the genus Achaetomiella, the genusAchaetomium, the genus Thielabia the genus Chaetomium, the genusGelasinospora, the genus Microascus, the genus Coniochaeta, or the genusEupenicillium (EP 1,291,416) and reacts with1-deoxyfructosyl-L-valyl-L-histidine (hereinafter also referred to asFVH), which have the above-described property, have been reported inrecent years.

However, a ketoamine oxidase belonging to 1) has the property (K4) and aketoamine oxidase belonging to 2) has the property (K2), but there is nodescription that they react with FVH. A ketoamine oxidase belonging to3), even a ketoamine oxidase that is derived from a microorganismbelonging to the genus Eupenicillium and reacts with FK at the most lowrate, reacts with FK at a rate of 9.78% in the case where the reactionwith FVH is defined as 100% (EP 1,291,416). Therefore, it is consideredthat the ketoamine oxidase sufficiently reacts with a glycated aminoacid and/or a glycated peptide from a site including intramolecularlysine cleaved from glycated hemoglobin by a protease, and there is nodescription about a reaction with a glycated peptide derived from aglycated α-chain N-terminal cleaved from glycated hemoglobin by aprotease, for example, 1-deoxyfructosyl-L-valyl-L-leucine (hereinafteralso referred to as FVL), so that it is not considered to be a ketoamineoxidase having the property (K1), (K2), or (K3).

As described above, there have not been reported ketoamine oxidasesthat: have the property (K1); have the property (K2) and reacts withFVH; or have the property (K3).

Meanwhile, in order to prepare ketoamine oxidases that have the property(K1), and have the property (K2) and reacts with FVH, it is consideredto perform modification of known ketoamine oxidases by amino acidsubstitution, deletion, insertion, etc. However, it has not been knownwhich amino acid residue in the primary structure of the enzymecontributes to reduction of a reaction with FK or FZK. Therefore, theactivity to FK or ε-1-deoxyfructosyl-(α-benzyloxycarbonyl-L-lysine)(hereinafter also referred to as FZK) cannot be reduced by modificationof any ketoamine oxidase gene, i.e., there has not been knownpreparation of ketoamine oxidases that have the property (K1), and havethe property (K2) and reacts with FVH by modification.

Meanwhile, there has not been known reduction of a rate of the activityto FK or FZK compared to that to FVH by regulating a reaction conditionfor a ketoamine oxidase capable of reacting with FVH.

In order to clearly distinguish and determine glycation of an α-aminogroup of valine in a β-chain N-terminal existing in glycated hemoglobinusing a protease and a ketoamine oxidase, the specificity of theprotease and ketoamine oxidase must be combined as described above.However, in JP-A-08-336386, WO 97/13872, JP-A-2001-95598, and ClinicalChemistry 49(2): 269-274 (2003), there is no description about specificdetermination of a glycated β-chain N-terminal of hemoglobin, and thereis only a description that the value that was obtained or may beobtained by the HPLC method significantly correlates with the determinedvalue obtained by the disclosed enzymatic method. Moreover, there is nomention about specificity of the used protease and ketoamine oxidase,and a glycated amino acid and/or a glycated peptide cleaved simply bydegrading glycated hemoglobin by a protease is detected by a ketoamineoxidase, so that it is considered that there was detected a mixture ofhemoglobin where an ε-amino group of intramolecular lysine and α-aminogroups of valine in α- and β-chain N-terminals have been glycated.Furthermore, in examples in JP-A-2001-95598, glycated hemoglobin wasdetermined using a protease and a ketoamine oxidase that reacts withFVH. However, centrifugation was performed as an operation, and there isno description that the determination can be performed without aseparation operation.

JP-A-2000-300294 suggests an enzymatic method of specificallydetermining only hemoglobin in which an α-amino group of valine inhemoglobin β-chain N-terminals has been glycated. In this method,sequential processing was performed by a protease capable of cleavingthe carboxyl group side of leucine at the third position from ahemoglobin β-chain N-terminal and then by a protease capable of cleavingHis-Leu from fructosyl-Val-His-Leu, to thereby generate fructosylvaline, and the amount of glycation of an α-amino groups of valine in ahemoglobin β-chain N-terminal was specifically determined. However, thismethod have the following disadvantages: it requires two stages ofprotease reactions; it is difficult to strictly control the proteasereaction for cleaving the carboxyl group side of leucine at the thirdposition from a hemoglobin β-chain N-terminal in the first stage; and areaction of the step for cleaving an α-glycated amino acid from ahemoglobin β-chain N-terminal glycated peptide in the second stagehardly proceeds in general and must be performed using a large amount ofenzymes for a long time. Moreover, in the method shown in examples, acumbersome separation operation (ultrafiltration) was performed twice,and there is no description that the method of the present applicationenables specific determination of a glycated β-chain N-terminal ofglycated hemoglobin without a separation operation.

EP 1,291,416 suggests a method of determining a glycated protein such ashemoglobin A1c with an oxidase capable of reacting with FVH to bereleased by a protease such as Molsin, AO-protease, Peptidase (availablefrom Kikkoman Corporation), carboxypeptidase Y, or Protin P (availablefrom Daiwa Kasei K.K.). The description further suggests, in the casewhere FK generated by a protease is problematic, determination by anoxidase capable of reacting with FVH after elimination of FK by afructosyl amine oxidase that reacts with FK, or determination using anoxidase that reacts FVH and hardly reacts with FK. However, there is nomention about distinction between a glycated amino acid and/or aglycated peptide derived from a glycated α-chain N-terminal of glycatedhemoglobin, and there are not demonstrated examples on determination ofa glycated β-chain N-terminal of glycated hemoglobin by the suggestedmethod. In addition, there is no description that the suggested methodcan be performed without a separation operation.

Although the above-described determination methods relating to glycatedhemoglobin were known, there have not been known a method and a reagentkit for specifically determining a glycated β-chain N-terminal ofglycated hemoglobin using enzymes without a separation operation.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method capable ofspecifically determining only a glycated β-chain N-terminal of glycatedhemoglobin without a separation operation, a determination reagent kit,a protease that can be used for the specific determination, a productionmethod thereof, a screening method thereof, and a ketoamine oxidase thatcan be used for the specific determination.

The inventors of the present invention have made extensive studies andas a result, firstly, we have invented a method of screening a proteasethat cleaves a glycated amino acid and/or a glycated peptide in aβ-chain N-terminal without substantially cleaving a glycated amino acidor a glycated peptide in an α-chain N-terminal of glycated hemoglobin ora fragment thereof. That is, we have invented a method of detecting theactivity of a protease that cleaves a glycated amino acid and/or aglycated peptide, which serves as a substrate for a ketoamine oxidase,from a glycated β-chain N-terminal without substantially cleaving aglycated amino acid or a glycated peptide, which serves as a substratefor a ketoamine oxidase, from a glycated α-chain N-terminal of glycatedhemoglobin by using glycated peptides, which serve as substrates for aprotease, existing in the α-chain N-terminal and β-chain N-terminal ofglycated hemoglobin.

Next, the invented screening method is used for screening of a widevariety of proteases that cleave a glycated amino acid and/or a glycatedpeptide, which serves as a substrate for a ketoamine oxidase, from aβ-chain N-terminal glycated pentapeptide without substantially cleavinga glycated amino acid or a glycated peptide, which serves as a substratefor a ketoamine oxidase, from an α-chain N-terminal glycatedpentapeptide. As a result, it has been found out that the proteasereaction of interest occurs in a commercially-available proteasepreparation such as a neutral proteinase derived from Bacillus sp.(manufactured by Toyobo Co., Ltd.) or in a protease produced by abacterium such as Bacillus sp., Aeromonas hydrophila, or Lysobacterenzymogenes.

In addition, of those proteases, the proteases produced by Aeromonashydrophila and Lysobacter enzymogenes have found to have homology toknown elastases. Conventionally, it is known that an elastase hassubstrate specificity and is known as an enzyme that cleaves a peptidebond on the C-terminal side of leucine, isoleucine, valine, or alanine.The present invention revealed for the first time that the elastasecleaves a peptide bond not on the C-terminal side but on the N-terminalside of leucine in1-deoxyfructosyl-L-valyl-L-histidyl-L-leucyl-L-threonyl-L-proline (SEQID NO: 36) (hereinafter also referred to as hemoglobin β-chainN-terminal glycated pentapeptide) to release FVH.

Next, there has been completed a method of specifically determining aglycated β-chain N-terminal of glycated hemoglobin or a fragment thereofusing enzymes without a separation operation by previously eliminatingFK and/or a glycated peptide including FK by a ketoamine oxidase thatdoes not react with a glycated peptide derived from a glycated β-chainN-terminal and reacts with FK or a glycated peptide including FK, in thecase where the protease of interest cleaves not only a glycated aminoacid and/or a glycated peptide from a glycated β-chain N-terminal butalso FK and/or a glycated peptide including FK from glycated hemoglobinor a fragment thereof without cleaving a glycated amino acid or aglycated peptide from a glycated α-chain N-terminal of glycatedhemoglobin or a fragment thereof.

Moreover, there has been found out a reaction condition capable ofsignificantly reducing reactivity to FZK of a ketoamine oxidase in thecase where the protease cleaves not only a glycated amino acid and/or aglycated peptide from a glycated β-chain N-terminal but also FK and/or apeptide including FK from glycated hemoglobin or a fragment thereof,without cleaving a glycated amino acid or a glycated peptide from aglycated α-chain N-terminal of glycated hemoglobin or a fragmentthereof. Meanwhile, preparation and use of a novel ketoamine oxidasehaving high specificity, which reacts with FZK at a rate of 5% or lessin the case where the reaction with FVH is defined as 100%, enabledspecific determination of only a glycated amino acid and/or a glycatedpeptide cleaved from a glycated β-chain N-terminal. That is, theinventors of the present invention have discovered that substitution ofamino acids at position 58 and 62 in a ketoanmine oxidase gene derivedfrom Curvularia clavata contributes to reduction of reactivity to FZK.Preparation and use of the oxidase based on such fact led to completionof a method of specifically determining a glycated β-chain N-terminal ofglycated hemoglobin or a fragment thereof using enzymes without aseparation operation.

Furthermore, the following reaction conditions for the protease ofinterest for degradation of glycated hemoglobin or a fragment thereofwere found out:

i) a condition not to cleave FK and/or a glycated peptide including FK,that can react with a ketoamine oxidase to be used in combination withthe protease of interest;

ii) a condition not to cleave a glycated amino acid or a glycatedpeptide that is derived from a glycated α-chain N-terminal and can reactwith a keto amine oxidase to be used in combination with the protease ofinterest; and

iii) a condition to cleave a glycated amino acid or a glycated peptidethat is derived from a glycated β-chain N-terminal and can react with aketoamine oxidase to be used in combination with the protease ofinterest. As the results, the inventors of the present invention havecompleted a method of specifically determining a glycated β-chainN-terminal of glycated hemoglobin or a fragment thereof using enzymeswithout separation operation.

That is, the present invention has the following configurations.

(1) A protease which cleaves a glycated amino acid and/or a glycatedpeptide from a glycated β-chain N-terminal of glycated hemoglobinwithout substantially cleaving a glycated amino acid or a glycatedpeptide from a glycated α-chain N-terminal of glycated hemoglobin.

(2) A protease which cleaves a glycated amino acid and/or a glycatedpeptide from a fragment including a glycated β-chain N-terminal ofglycated hemoglobin without substantially cleaving a glycated amino acidor a glycated peptide from a fragment including a glycated α-chainN-terminal of glycated hemoglobin.

(3) A protease whose reaction to cleave a glycated amino acid or aglycated peptide from a glycated α-chain N-terminal of glycatedhemoglobin occurs at 10% or less in the case where a reaction to cleavea glycated amino acid and/or a glycated peptide from a glycated β-chainN-terminal of glycated hemoglobin is defined as 100%.

(4) A protease according to any one of the above items (1) to (3), inwhich a glycated peptide cleaved from a glycated β-chain N-terminal ofglycated hemoglobin or a fragment including a glycated β-chainN-terminal of glycated hemoglobin is1-deoxyfructosyl-L-valyl-L-histidine.

(5) A protease according to any one of the above items (1) to (4), inwhich the glycated amino acid and glycated peptide that are notsubstantially cleaved from the glycated α-chain N-terminal of glycatedhemoglobin or the fragment including the glycated α-chain N-terminal ofglycated hemoglobin are 1-deoxyfructosyl-L-valine and1-deoxyfructosyl-L-valyl-L-leucine, respectively.

(6) A protease according to any one of the above items (1) to (5), whichis a protease derived from a bacterium belonging to the genusLysobacter.

(7) A protease according to any one of the above items (1) to (5), whichis a protease derived from Bacillus sp. ASP-842 (FERM BP-08641) orAeromonas hydrophila NBRC 3820.

(8) A protease according to any one of the above items (1) to (7), whichis a metalloprotease, neutral protease, or elastase.

(9) An elastase which cuts a peptide bond on the N-terminal side ofleucine in a protein or peptide.

(10) A Lysobacter enzymogenes YK-366 (FERM BP-10010) strain.

(11) A Bacillus sp. ASP-842 (FERM BP-08641) strain.

(12) A method of producing a protease according to the above item (6),which includes the following steps (a) and (b):

(a) culturing a bacterium belonging to the genus Lysobacter in a culturesolution; and

(b) extracting a protease from the culture solution.

(13) A method of producing the protease according to the above item (7),which includes the following steps (a) and (b):

(a) culturing Bacillus sp. ASP-842 (FERM BP-08641) or Aeromonashydrophila NBRC 3820 in a culture solution; and

(b) extracting a protease from the culture solution.

(14) A ketoamine oxidase which has the following property (A):

(A) the reactivity to ε-1-deoxyfructosyl-(α-benzyloxycarbonyl-L-lysine)is 5% or less than that to 1-deoxyfructosyl-L-valyl-L-histidine.

(15) A ketoamine oxidase according to the above item (14), which furtherhas the following properties (B) and (C):

(B) consisting of amino acids having at least 75% homology to the aminoacid sequence described in SEQ ID NO: 1; and

(C) at least an amino acid at position 58 or 62 in the amino acidsequence described in SEQ ID NO: 1 being substituted by another aminoacid.

(16) A ketoamine oxidase according to the above item (15), in which inthe amino acid substitution described in (C), the amino acid at position58 is substituted by valine, threonine, asparagine, cysteine, serine, oralanine; and the amino acid at position 62 is substituted by histidine.

(17) A gene which encodes an amino acid sequence of a ketoamine oxidaseaccording to any one of the above items (14) to (16).

(18) A ketoamine oxidase-expression vector which contains a geneaccording to the above item (17).

(19) A host cell which contains an expression vector according to theabove item (18).

(20) A method of specifically determining a glycated β-chain N-terminalof glycated hemoglobin using enzymes without a separation operation.

(21) A determination method according to the above item (20), in whichthe enzymes include a protease (i).

(22) A determination method according to the above item (21), in whichthe enzymes further include a ketoamine oxidase (ii).

(23) A determination method according to the above item (22), in whichthe N-terminal is specifically determined via the following reactionsteps (iii) and/or (iv):

(iii) a reaction step wherein the protease (i) cleaves a glycated aminoacid and/or a glycated peptide from a glycated β-chain N-terminal ofglycated hemoglobin without substantially cleavingε-1-deoxyfructosyl-L-lysine and/or a glycated peptide includingε-1-deoxyfructosyl-L-lysine, which the ketoamine oxidase (ii) reactswith, from glycated hemoglobin; and

(iv) a reaction step in which the reactivity of the ketoamine oxidase(ii) to ε-1-deoxyfructosyl-(α-benzyloxycarbonyl-L-lysine) is 30% or lesscompared with that to 1-deoxyfructosyl-L-valyl-L-histidine.

(24) A determination method according to the above item (23), in whichthe reaction step (iii) is performed under a reaction condition of pH5.0 to 6.0.

(25) A determination method according to the above item (23) or (24), inwhich the reaction step (iv) is performed under a reaction condition ofpH 5.5 to 6.5.

(26) A determination method according to any one of the above items (21)to (25), in which the protease (i) is a protease according to any one ofthe above items (1) to (8).

(27) A determination method according to any one of the above items (22)to (26), in which the ketoamine oxidase (ii) is a ketoamine oxidase thatreacts with a glycated amino acid and/or a glycated peptide each cleavedfrom a glycated β-chain N-terminal of glycated hemoglobin by a protease.

(28) A determination method according to the above item (27), in whichthe ketoamine oxidase is derived from a bacterium belonging to the genusCurvularia.

(29) A determination method according to any one of the above items (22)to (28), in which the ketoamine oxidase (ii) includes the following twokinds of ketoamine oxidases (a) and (b):

(a) a ketoamine oxidase that reacts with a glycated amino acid and/or aglycated peptide each cleaved from a glycated β-chain N-terminal ofglycated hemoglobin by a protease; and

(b) a ketoamine oxidase that reacts with ε-1-deoxyfructosyl-L-lysineand/or a glycated peptide including ε-1-deoxyfructosyl-L-lysine withoutsubstantially reacting with a glycated amino acid and/or a glycatedpeptide each cleaved from a glycated β-chain N-terminal of glycatedhemoglobin by a protease.

(30) A determination method according to the above item (29), in whichthe ketoamine oxidase (a) is derived from a bacterium belonging to thegenus Curvularia and/or the ketoamine oxidase (b) is derived from abacterium belonging to the genus Fusarium.

(31) A determination method according to any one of the above items (22)to (26), in which the ketoamine oxidase (ii) is a ketoamine oxidaseaccording to any one of the above items (14) to (16).

(32) A method of screening a protease that cleaves a glycated amino acidand/or a glycated peptide from a glycated β-chain N-terminal withoutsubstantially cleaving a glycated amino acid or a glycated peptide froma glycated α-chain N-terminal of glycated hemoglobin or a fragmentthereof, in which a hemoglobin α-chain N-terminal glycated peptidehaving a length of 3 amino acids to 20 amino acids and a hemoglobinβ-chain N-terminal glycated peptide having a length of 3 amino acids to20 amino acids are used.

(33) A screening method according to the above item (32), in which thehemoglobin α-chain N-terminal glycated peptide and hemoglobin β-chainN-terminal glycated peptide have a length of 5 amino acids.

(34) A reagent kit for specifically determining a glycated β-chainN-terminal of glycated hemoglobin without a separation operation, whichincludes the following (i) and (ii):

(i) a protease; and

(ii) a ketoamine oxidase according to the above item (14).

According to the present invention, there are provided a method capableof specifically determining only a glycated β-chain N-terminal ofglycated hemoglobin without a separation operation, a determinationreagent kit, a protease to be used for the specific determination, aproduction method or a screening method for the protease, and aketoamine oxidase that can be used for the specific determination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the homology in amino acid sequences of ketoamine oxidases(SEQ ID NOS 33, 34, 32 & 35, disclosed respectively in order ofappearance).

FIG. 2 shows the optimum pH for a protease derived from Bacillus sp. ASP842.

FIG. 3 shows the pH stability of a protease derived from Bacillus sp.ASP 842.

FIG. 4 shows the optimum temperature for a protease derived fromBacillus sp. ASP 842.

FIG. 5 shows the thermal stability of a protease derived from Bacillussp. ASP 842.

FIG. 6 shows the optimum pH for a protease derived from Aeromonashydrophila NBRC 3820.

FIG. 7 shows the pH stability of a protease derived from Aeromonashydrophila NBRC 3820.

FIG. 8 shows the optimum temperature for a protease derived fromAeromonas hydrophila NBRC 3820.

FIG. 9 shows the thermal stability of a protease derived from Aeromonashydrophila NBRC 3820.

FIG. 10 shows the optimum pH for a protease derived from Lysobacterenzymogenes YK-366.

FIG. 11 shows the pH stability of a protease derived from Lysobacterenzymogenes YK-366.

FIG. 12 shows the optimum temperature for a protease derived fromLysobacter enzymogenes YK-366.

FIG. 13 shows the thermal stability of a protease derived fromLysobacter enzymogenes YK-366.

FIG. 14 shows a restriction map of a plasmid pPOSFOD923.

FIG. 15 shows the relationship between an absorbance difference ΔAs andan FVH concentration.

FIG. 16 shows the relationship between a protease reaction time and aresultant determined value.

FIG. 17 shows a determination scheme in the case where an FVH amount isdetermined using FOD923 after elimination of signals derived from aglycated lysine and a peptide including a glycated lysine with FOD2after degradation of glycated hemoglobin by a protease.

FIG. 18 shows a determination scheme in the case where an FVH amount isdetermined using FOD923 without elimination of signals derived from aglycated lysine and a peptide including a glycated lysine afterdegradation of glycated hemoglobin by a protease.

FIG. 19 shows a determination scheme in the case where an FVH amount isdetermined using FOD923M without elimination of signals derived from aglycated lysine and a peptide including a glycated lysine afterdegradation of glycated hemoglobin by a protease.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, configurations and preferred modes of the present inventionwill be described in more detail.

<Amadori Compound>

The term “Amadori compound” in the present invention refers to acompound having a ketoamine structure represented by a general formula—(CO)—CHR—NH— (R represents a hydrogen atom or a hydroxyl group), whichis generated by a Maillard reaction of a compound having an amino groupsuch as a protein and a compound having an aldehyde group such asglucose. The Amadori compound includes: a glycated protein such asglycated hemoglobin or glycated albumin; a glycated peptide generated byglycation of a peptide; etc.

<Glycated Hemoglobin>

The term refers to an Amadori compound generated by glycation ofhemoglobin by a Maillard reaction, in which each α-amino group of valinein α-chain and β-chain N-terminals or an ε-amino group of intramolecularlysine is considered to be glycated. The fragment of glycated hemoglobinrefers to a peptide obtained by degradation of glycated hemoglobin.

<Hemoglobin A1c>

In a definition accepted as the international standard, hemoglobin A1cis believed to be glycated hemoglobin in which an α-amino group ofvaline in a hemoglobin β-chain N-terminal is glycated (ClinicalChemistry and Laboratory Medicine 36(5): 299-308 (1998)).

<Specifically>

The phrase “specifically determining a glycated β-chain N-terminal ofglycated hemoglobin” is used in the case where a value obtained bydetermination of a glycation level of glycated hemoglobin is derivedfrom glycation of an α-amino group of valine in a hemoglobin β-chainN-terminal at a rate of 80% or more, desirably 90% or more, moredesirably 95% or more.

<Glycated Amino Acid and Glycated Peptide>

In the phrase “without a protease substantially cleaving a glycatedamino acid or a glycated peptide from a glycated α-chain N-terminal ofglycated hemoglobin or a fragment thereof”, the term “glycated aminoacid” refers to 1-deoxyfructosyl-L-valine, while the term “glycatedpeptide” refers to a peptide that has a length of 20 amino acids or lessfrom the α-chain N-terminal of hemoglobin, includes1-deoxyfructosyl-L-valine as an N-terminal valine, and is detected by aketoamine oxidase to be used in combination with a protease. Forexample, in the case where a ketoamine oxidase derived from Curvulariaclavata YH 923 (FERM BP-10009) is used in combination with a protease,the term refers to 1-deoxyfructosyl-L-valyl-L-leucine.

In the phrase “a protease cleaves a glycated amino acid or a glycatedpeptide from a glycated β-chain N-terminal of glycated hemoglobin or afragment thereof”, the term “glycated amino acid” refers to1-deoxyfructosyl-L-valine, while the term “glycated peptide” refers to apeptide that has a length of 20 amino acids or less from the β-chainN-terminal of hemoglobin, includes 1-deoxyfructosyl-L-valine as anN-terminal valine, and is detected by a ketoamine oxidase to be used incombination with a protease. For example, in the case where a ketoamineoxidase derived from Curvularia clavata YH 923 (FERM BP-10009) is usedin combination with a protease, the term refers to1-deoxyfructosyl-L-valyl-L-histidine. In the phrase “a protease cleavesa glycated peptide including ε-1-deoxyfructosyl-L-lysine from glycatedhemoglobin”, the term “glycated peptide” refers to a glycated peptidehaving a length of 50 amino acids or less.

Note that, Curvularia clavata YH 923 (FERM BP-10009) has been depositedin International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan on Feb. 12, 2003.

<Substantially>

In a protease reaction, the phrase “cleaving a glycated amino acidand/or a glycated peptide from a glycated β-chain N-terminal withoutsubstantially cleaving a glycated amino acid or a glycated peptide froma glycated α-chain N-terminal” refers to the fact that the reactivity tocleave a glycated amino acid or a glycated peptide from a glycatedα-chain N-terminal is 10% or less, desirably 1% or less, more desirably0.1% or less in the case where the reactivity to cleave a glycated aminoacid and/or a glycated peptide from a glycated β-chain N-terminal isdefined as 100%. Note that the above-described protease reaction isdetermined by detecting cleaved products using a ketoamine oxidase,preferably a ketoamine oxidase derived from Curvularia clavata YH 923(FERM BP-10009) or a ketoamine oxidase derived from Fusarium oxysporum(manufactured by Asahi Kasei Pharma Corporation).

In a ketoamine oxidase reaction, the phrase “reacting with an ε-glycatedamino acid without substantially reacting with a glycated amino acidand/or a glycated peptide cleaved from a glycated β-chain N-terminal bya protease” refers to the fact that the reactivity to a glycated aminoacid and/or a glycated peptide cleaved from a glycated β-chainN-terminal by a protease is 10% or less, preferably 8% or less, morepreferably 1% or less, most preferably 0.1% or less in the case wherethe reactivity to an ε-glycated amino acid is defined as 100%.Meanwhile, in a ketoamine oxidase reaction, the phrase “reacting with aglycated amino acid and/or a glycated peptide cleaved from a glycatedβ-chain N-terminal by a protease without substantially reacting with anε-glycated amino acid” refers to the fact that the reactivity to anε-glycated amino acid is 8% or less, desirably 1% or less, moredesirably 0.1% or less in the case where the reactivity to a glycatedamino acid and/or a glycated peptide cleaved from a glycated β-chainN-terminal by a protease is defined as 100%. Note that the ketoamineoxidase reaction is determined by detecting generated hydrogen peroxide.

<Ketoamine Oxidase>

The term refers to an enzyme that reacts with a compound having aketoamine structure to generate hydrogen peroxide, which is also knownas fructosyl-amine oxidase.

<Ketoamine Oxidase having Reactivity toε-1-deoxyfructosyl-(α-benzyloxycarbonyl-L-lysine) of 5% or less Comparedwith Reactivity to 1-deoxyfructosyl-L-valyl-L-histidine>

The phrase “reactivity of a ketoamine oxidase toε-1-deoxyfructosyl-(α-benzyloxycarbonyl-L-lysine) (hereinafter alsoreferred to as FZK) is 5% or less compared with that to1-deoxyfructosyl-L-valyl-L-histidine (hereinafter also referred to asFVH)” refers to the fact that the rate of the reactivity is 5% or less,which is determined by the following steps: adding 20 μl of a ketoamineoxidase solution to 200 μl of a reaction solution (50 mM Tris-HCl buffer(pH 7.5), 0.1% Triton X-100, 0.03% 4-aminoantipyrine, 0.02% TOOS, 5 U/mlperoxidase, and 2 mM FZK or FVH); allowing the mixture to react at 37°C. for 5 minutes; adding 0.5 ml of 0.5% SDS; determining an absorbanceat 555 nm (A1); performing the same operations using a reaction solutioncontaining no FZK and FVH to determine an absorbance (Ab); anddetermining the reactivity from the difference (A1−Ab).

<Separation Operation>

The term “separation operation” refers to an operation for increasingthe purity or concentration of glycated hemoglobin or a substancederived from glycated hemoglobin, in a process of a series ofdetermination steps for determining glycated hemoglobin in a sample suchas a whole blood specimen or hemolyzed red blood cell specimen, whichincludes column chromatography operation, membrane filtration operation,adsorption separation operation, and precipitate separation operation.

In order to specifically determine a glycated β-chain N-terminal ofglycated hemoglobin using enzymes without a separation operation, amongthree kinds of glycated sites in glycated hemoglobin, intramolecularlysine, α-chain N-terminal, and β-chain N-terminal, the enzymes musthave specificity only to the glycated site of the β-chain N-terminal. Asenzymes to be used for obtaining such specificity, one kind of enzymemay be used, or plural kinds of enzymes may be used in combination. Forexample, the specificity may be obtained by combining oxidases,dehydrogenases, kinases, etc., which are enzymes that react with aglycated amino acid and/or a glycated peptide derived from a glycatedβ-chain N-terminal cleaved by degradation of glycated hemoglobin by aprotease. Moreover, in order to determine only a β-chain N-terminalglycated using a protease or a ketoamine oxidase, it is necessary thatthe enzymes be combined according to the specificity as below.

That is, in the case where proteases and ketoamine oxidases areclassified into (P1) to (P4) and (K1) to (K5), respectively, it isnecessary that those enzymes be combined as below: <(P1) and (K1) or(K2) or (K3) or (K4)>, <(P2) and (K1) or (K3)>, <(P3) and (K1) or (K2)>,<(P4) and (K1)>, <(P3) and (K5) and (K3)>, and <(P3) and (K5) and (K4)>.

The properties of the respective classified enzymes are as follows.

(P1) cleaves a glycated amino acid and/or a glycated peptide only from aglycated β-chain N-terminal of glycated hemoglobin,

(P2) cleaves a glycated amino acid and/or a glycated peptide only fromglycated α- and β-chain N-terminals of glycated hemoglobin,

(P3) cleaves a glycated amino acid and/or a glycated peptide only from aglycated β-chain N-terminal of glycated hemoglobin and a site includinga intramolecular lysine,

(P4) cleaves a glycated amino acid and/or a glycated peptide from aglycated α- and β-chain N-terminals of glycated hemoglobin and a siteincluding a intramolecular lysine,

(K1) reacts only with a glycated amino acid and/or a glycated peptidederived from a glycated β-chain N-terminal of glycated amino acidsand/or glycated peptides cleaved from glycated hemoglobin by a proteaseto be used in combination,

(K2) reacts only with a glycated amino acid and/or a glycated peptidederived from glycated α- and β-chain N-terminals of glycated amino acidsand/or glycated peptides cleaved from glycated hemoglobin by a proteaseto be used in combination,

(K3) reacts only with a glycated amino acid and/or a glycated peptidederived from a glycated β-chain N-terminal and from a site includingintramolecular lysine of glycated amino acids and/or glycated peptidescleaved from glycated hemoglobin by a protease to be used incombination,

(K4) reacts with a glycated amino acid and/or a glycated peptide derivedfrom glycated α- and β-chain N-terminals and from a site includingintramolecular lysine that have been cleaved from glycated hemoglobin bya protease to be used in combination, and

(K5) reacts with a glycated amino acid and/or a glycated peptide from asite including intramolecular lysine without reacting with a glycatedamino acid and/or a glycated peptide derived from a glycated β-chainN-terminal of glycated amino acids and/or glycated peptides cleaved fromglycated hemoglobin by a protease to be used in combination.

<Screening of Protease>

The inventors of the present invention have made extensive studies andas a result have invented, as a screening method of obtaining a proteasehaving the specificity (P1) or (P3), a method of selecting a protease tocleave a glycated amino acid and/or a glycated peptide only when ahemoglobin β-chain N-terminal glycated peptide is used as a substrate inthe case of using a hemoglobin α-chain N-terminal glycated peptide and ahemoglobin β-chain N-terminal glycated peptide as substrates. Thelengths of the α-chain N-terminal glycated peptide and β-chainN-terminal glycated peptide used above are not particularly limited, butit has been found out that screening may be performed using a glycatedpeptide having a length of 3 amino acids to 20 amino acids. The peptidesmay be derived from chemically synthesized compounds or natural productssuch as glycated hemoglobin.

Specific examples thereof include: as the β-chain N-terminal glycatedpeptide,1-deoxyfructosyl-L-valyl-L-histidyl-L-leucyl-L-threonyl-L-proline (SEQID NO: 36) (manufactured by Peptide Institute, Inc., hereinafter alsoreferred to as β-glycated pentapeptide); and as the α-chain N-terminalglycated peptide,1-deoxyfructosyl-L-valyl-L-leucyl-L-seryl-L-prolyl-L-alanine (SEQ ID NO:50) (manufactured by Peptide Institute, Inc., hereinafter also referredto as α-glycated pentapeptide). In the case where those pentapeptidesare used as substrates, a glycated amino acid, glycated dipeptide,glycated tripeptide, or glycated tetrapeptide may be cleaved by aprotease. A glycated amino acid and/or a glycated peptide cleaved froman α-chain N-terminal glycated peptide and a β-chain N-terminal glycatedpeptide by a protease may be detected using an enzyme that reacts withthe cleaved glycated amino acid and/or glycated peptide withoutsubstantially reacting with an α-chain N-terminal glycated peptide and aβ-chain N-terminal glycated peptide, and examples of such enzyme includeoxidases, dehydrogenases, and kinases.

An example of the oxidase includes ketoamine oxidase. Examples of theketoamine oxidase include oxidases derived from the genus Achaetomiella,the genus Achaetomium, the genus Thielavia, the genus Chaetomium, thegenus Gelasinospora, the genus Microascus, the genus Coniochaeta, thegenus Eupenicillium (all of them are described in the description of EP1,291,416), the genus Corynebacterium (JP-A-61-268178), the genusAspergillus (JP-A-03-155780), the genus Penicillium (JP-A-04-4874), thegenus Fusarium (JP-A-05-192193, JP-A-07-289253, JP-A-08-154672), thegenus Gibberella (JP-A-05-192153, JP-A-07-289253), the genus Candida(JP-A-0646846), the genus Aspergillus (JP-A-10-33177, JP-A-10-33180),Neocosmospora vasinfecta (NBRC 7590), Coniochaetidium savoryi(ATCC36547), Arthrinium sp. TO6 (FERM P-19211), Arthrinium phaeospermum(NBRC 31950), Arthrinium phaeospermum (NBRC 6620), Arthrinium japonicam(NBRC 31098), Pyrenochaeta sp. YH807 (FERM P-19210), Pyrenochaetagentianicola (MAFF425531), Pyrenochaeta terrestris (NBRC 30929),Leptosphaeria nodorum (name during the conidial generation is Phomahennebelgii) (NBRC 7480), Leptosphaeria doliolum (JCM2742),Leptosphaeria maculans (name during the conidial generation is Phomalingam) (MAFF726528), Pleospora herbarum (NBRC 32012), Pleospora betae(name during the conidial generation is Phoma betae) (NBRC 5918),Ophiobolus herpotrichus (NBRC 6158), Curvularia clavata YH923 (FERMBP-10009), and mutant enzymes thereof.

The substrate specificities of a ketoamine oxidase derived fromCurvularia clavata YH 923 (FERM BP-10009) to FVH is 100%, while that to1-deoxyfructosyl-L-valyl-L-histidyl-L-leucine (hereinafter also referredto as β-glycated tripeptide or FVHL),1-deoxyfructosyl-L-valyl-L-histidyl-L-leucyl-L-threonine (SEQ ID NO: 37)(hereinafter also referred to as β-glycated tetrapeptide or FVHLT),β-glycated pentapeptide, 1-deoxyfructosyl-L-valyl-L-leucine (hereinafteralso referred to as FVL) and α-glycated pentapeptide are 0.09%, 0.0009%,0%, 3.4%, and 0.01%, respectively. Therefore, the activity of a proteaseto cleave FVH from a β-glycated pentapeptide and the activity of aprotease to cleave FVL from an α-glycated pentapeptide may be detectedby using the ketoamine oxidase. In the case where a glycated amino acidand/or a glycated peptide cleaved by a protease are detected using anoxidase, the amount of generated hydrogen peroxide may be detected byelectrodes, luminescence, fluorescence, absorbance, etc, but it is easyto detect absorbance using peroxidase and a chromogen. For example,protease activity may easily be confirmed by determining absorbancechanges from at 540 to 570 nm using 4-aminoantipyrine (manufactured byWako Pure Chemical Industries, Ltd.) and TODB (manufactured by DojindoLaboratories) as chromogens.

Examples of a protease that is obtained by the above-described screeningmethod and has the specificity (P1) or (P3) include proteases derivedfrom bacteria belonging to the genus Bacillus, the genus Aeromonas, andthe genus Lysobacter. More specific examples thereof include: neutralproteinase derived from Bacillus sp. (manufactured by Toyobo Co., Ltd.);and a protease derived from Bacillus sp. ASP 842 (FERM BP-08641),Aeromonas hydrophila NBRC 3820, or Lysobacter enzymogenes YK-366 (FERMBP-10010).

Of those strains, Bacillus sp. ASP 842 (FERM BP-08641) and Lysobacterenzymogenes YK-366 (FERM BP-10010) are novel strains that have beenisolated by the inventors of the present invention, and Bacillus sp. ASP842 (FERM BP-08641) and Lysobacter enzymogenes YK-366 (FERM BP-10010)have been deposited in International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology,Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan on Feb. 24,2004 and on Jan. 30, 2004, respectively.

The mycological properties of those deposited strains are shown belowand were identified as follows.

1. ASP842 (FERM BP-08641) is a Gram-positive bacillus having a size of0.8 to 1.0×2.0 to 3.0 μm and has the properties shown in Table 1, sothat it was identified as Bacillus sp. by Bergy's Manual of SystematicBacteriology (1984).

TABLE 1 (a) Morphological Properties Culture condition (Medium: NutrientAgar; 30 degree C.) Cell shape 0.8-1.0 × 2.0-3.0 μm Cell polymorphism −Motility + (peritrichous) Sporulation + (b) Cultural Properties Culturecondition (Medium: Nutrient Agar; 30 degree C.) Color creamy Gloss −Pigment production − Culture condition (Liquid medium: Nutrient Broth;30 degree C.) Growth on the surface + Opacity of medium − Culturecondition (gelatin stab culture; 30 degree C.) Growth + Gelatinliquefaction + Culture codintion (litmus milk; 30 degree C.) coagulation− liquefaction − (c) Physiological Properties  1) Gram staining +  2)Nitrate Reduction +  3) Denitrification Reaction −  4) MR test −  5) VPtest +  6) Indole Production −  7) Hydrogen sulfide production −  8)Starch Hydrolysis +  9) Utilization of citrate (Koser/Christensen) −/+10) Utilization of inorganic nitorgen source −/+w   (nitrate/ammoniumsalt) 12) Urease activity − 13) Oxidase + 14) Catalase + 15) GrowthRange pH 5/8/10 +/+/+w   Growth Range Temperature 20/25/45/50 +/+/+/+16) Growth Condition (aerobic/anerobic) +/− 17) O-F Test(oxidation/fermentation) −/− 18) Acid/Gas production from sugarsL-arabinose −/− D-glucose +/− D-fructose +/− maltose +/− lactose +/−D-sorbitol +/− inositol +/− D-xylose +/− D-mannose +/− D-galactose −/−sucrose +/− trehalose +/− D-mannitol +/− glycerin +/− (d) Otherphysiological properties beta-galactosidase activity − argininedihydrolase activity − lysine decarboxylase activity − tryptophandeaminase activity + gelatinase activity + phosphatase activity +Propionate utilization − Growth in the presence of 10% NaCl + Caseinhydrolysis + Hippuric acid hydrolysis −

2. YK-366 (FERM BP-10010) is a Gram-negative aerobic bacillus having asize of 0.4×5 to 50 μm and has the properties shown in Table 2, so thatit was identified as Lysobacter enzymogenes by Bergy's Manual ofSystematic Bacteriology (1989).

TABLE 2 Tests Results Shape bacillus Gram Staining − Motility − Pigmentproduction* +, brown Colony texture mucoid Colony color pale yellowCatalase − Oxidase − VP reaction − Methyl red reaction − Indoleproduction − L-arginine Utilization − Urease − Hydorolysis esculin +gelatin + starch − beta-galactosidase + Growth +, pink on EMB agarmedium Assimilation of carbon sources glucose + L-arabinose −D-mannose + D-mannitol + N-acetyl-D-glucosamine − maltose + potassiumgluconate + n-caproic acid − adipic acid − DL-malic acid + sodiumcitrate − phenyl acetate − *PCA Plate Colony Count Agar<Production of Protease from Cultured Product>

Next, there will be described a method of culturing a protease-producingbacterium that may be used in the present invention and a method ofproducing the protease. As means for culturing the protease-producingbacterium of the present invention, solid culture or liquid culture maybe employed, but preferable is aeration liquid culture using a flask,jar fermenter, or the like. As a medium, a wide variety of media to begenerally used for culture of bacteria may be used. Examples of a carbonsource to be used include glucose, glycerol, sorbitol, lactose, mannose,or the like. Examples of a nitrogen source to be used include an yeastextract, a meat extract, tryptone, peptone, or the like. Examples of aninorganic salt to be used include sodium chloride, magnesium chloride,magnesium sulfate, calcium chloride, or the like. For cultureconditions, there may be employed a culture time such that the targetprotease gains the maximum titer or maximum purity at pH 5.0 to 8.0 andculture temperature of from 25 to 37° C., and for example, culture maybe performed for 16 to 72 hours.

Subsequently, collection of the protease will be described. In the casewhere the protease is secreted from cells, a solution containing a crudeprotease obtained by removing the cells from a culture medium byfiltration, centrifugation, etc is use. Meanwhile, in the case where theprotease is present in cells, there is used a solution containing acrude protease that is obtained by the following steps: separating thecells from a culture medium by filtration, centrifugation, etc.;suspending the cells in a buffer such as a phosphate buffer or Tris-HClbuffer supplemented with, if necessary, a surfactant, metallic salt,saccharide, amino acid, polyol, chelator, etc.; homogenizing the cellsusing lysozyme, ultrasonic waves, glass beads, etc.; and removinginsoluble products by filtration, centrifugation, etc. The solutioncontaining the crude protease is treated using a known isolation orpurification means for proteins or enzymes to yield a purified protease.For example, general enzyme purification methods such as a fractionalprecipitation method using an organic solvent including acetone orethanol, salting-out method using ammonium sulfate or the like,ion-exchange chromatography method, hydrophobic chromatography method,affinity chromatography method, and gel filtration method may beappropriately selected and combined, to thereby obtaining a purifiedprotease. The purified protease may be cryopreserved after addingappropriate stabilizer(s) such as sucrose, glycerol, or an amino acid(about 1 to 50%) and a coenzyme or the like (about 0.01 to 1%) singly orin combination of two or more of them.

<Screening of Ketoamine Oxidase>

Examples of a screening method for obtaining a ketoamine oxidase thathas the property (K1); has the property (K2) and reacts with FVH; or hasthe property (K3) include a method based on an index for reactivity to:FVL as a representative example of a glycated amino acid and/or aglycated peptide derived from an α-chain N-terminal; FVH as arepresentative example of a glycated amino acid and/or a glycatedpeptide derived from an β-chain N-terminal; or FK or ZFK as arepresentative example of a glycated amino acid and/or a glycatedpeptide from a site including intramolecular lysine, which have beencleaved from glycated hemoglobin by a protease. In particular, themethod is performed based on an index such that the reactivity to FK orFZK is 5% or less compared with that to FVH. The reactivity ispreferably 4% or less, more preferably 2% or less. The thus-obtainedketoamine oxidase that has the property (K1); has the property (K2) andreacts with FVH; or has the property (K3) may be: a natural ketoamineoxidase isolated from nature; a mutant ketoamine oxidase obtained byartificial modification of a natural ketoamine oxidase using a knowngenetic engineering technique such as amino acid substitution, deletion,or insertion; and ketoamine oxidase that may be obtained by chemicallymodifying natural and mutant ketoamine oxidases using a polyethyleneglycol derivative, succinimide derivative, maleimide derivative, etc.

<Mutated Ketoamine Oxidase>

No particular limitations are imposed on an existing ketoamine oxidasethat may be used in obtaining a mutated ketoamine oxidase that has aproperty described in (K1); has a property described in (K2) and reactswith FVH; or has a property described in (K3) by artificial modificationof one or more amino acids of an existing ketoamine oxidase such assubstitution, deletion, or insertion. Examples thereof include theketoamine oxidase for obtaining a protease having the specificity (P1)or (P3), which is described in the screening method.

Meanwhile, examples of a ketoamine oxidase that reacts with FVH includeketoamine oxidases derived from microorganisms belonging to the generaConiochaeta (EP 1,291,416), Eupenicillium (EP 1,291,416), Curvularia,and Neocosmospora.

Those enzymes have been analyzed on a genetic level, and the primarystructures have been estimated as shown in FIG. 1. The homology of thoseenzymes on an amino acid level is 75% or more, and there are conservedregions represented by the symbols “*”, so that those enzymes areincluded as examples of a ketoamine oxidase that includes an amino acidsequence having at least 75% of homology to the amino acid sequencedescribed in SEQ ID NO: 1. Meanwhile, substitution, deletion, orinsertion of an amino acid is not limited as long as mutation isperformed so as to have the property (K1); have the property (K2) andreact with FVH; or have the property (K3), but it is desirable that, forexample, at least one of amino acids corresponding to amino acids atpositions 58 and 62 in the amino acid sequence described in SEQ ID NO: 1be substituted. It is more desirable that an amino acid corresponding toan amino acid at position 58 be substituted by valine, threonine,asparagine, cysteine, serine, or alanine; and an amino acidcorresponding to an amino acid at position 62 be substituted byhistidine.

<Mutated Ketoamine Oxidase Expression Vector and Host Cell>

A ketoamine oxidase that has the property K1); has the property (K2) andreacts with FVH; or has the property (K3) may be obtained as follows: ifnecessary, a gene encoding the enzyme is ligated to a plasmid vector;the gene is transferred to a host microorganism such as a microorganismbelonging to the genus Saccharomyces, the genus Pichia, the genusAcremonium, the genus Bacillus, the genus Pseudomonas, the genusThermus, or the genus Escherichia; the microorganism is cultured;alternatively the encoding gene is transcribed and translated in vitro;and a treatment is performed using a known isolation or purificationmeans for proteins or enzymes.

<Determination Reagent Kit>

A method and reagent kit for specifically determining a glycated β-chainN-terminal of glycated hemoglobin using a protease and a ketoamineoxidase without a separation operation may include a combination of aprotease and a ketoamine oxidase to obtain specificity to a glycatedβ-chain N-terminal as well as a peroxidase and chromogen, and ifnecessary, there may be appropriately added a buffer component, salt,surfactant, metallic ion, electron acceptor, chelator, sugar, aminoacid, ascorbate oxidase, tetrazolium salt, polyol, enzyme stabilizer,enzyme reaction promoter, antibacterial agent, antioxidant, reductant,coenzyme, etc.

In the case where proteases and ketoamine oxidases are divided into (P1)to (P4) and (K1) to (K5), respectively, as described above, allcombinations of <(P1) and (K1) or (K2) or (K3) or (K4)>, <(P2) and (K1)or (K3)>, <(P3) and (K1) or (K2)>, <(P4) and (K1)>, <(P3) and (K5) and(K3)>, and <(P3) and (K5) and (K4)> may be employed as combinations of aprotease and a ketoamine oxidase to obtain specificity to a glycatedβ-chain N-terminal. However, in general, in the case where an N-terminalamino group of a protein has been glycated, reactivity for cleavage of aglycated amino acid from an N-terminal region is low. Therefore,desirable is a protease to cleave a glycated peptide from a glycatedamino acid in a β-chain N-terminal. Examples of a protease that isobtained by the above-described screening of a protease, has theproperty (P3), and has a property to cleave FVH from a β-chainN-terminal include proteases derived from bacteria belonging to thegenera Bacillus, Aeromonas, Lysobacter, etc. More specific examplesthereof include a neutral proteinase derived from Bacillus sp.(manufactured by Toyobo Co., Ltd.), proteases derived from Bacillus sp.ASP-842 (FERM BP-08641), Aeromonas hydrophila NBRC 3820, and Lysobacterenzymogenes YK-366 (FERM BP-10010). Meanwhile, in order to improve thereactivity while maintaining the specificity, the screened protease maybe used in combination with other appropriate protease.

The length of a glycated peptide cleaved from a glycated β-chainN-terminal of hemoglobin by a protease is 20 amino acids or less and isnot particularly limited as long as the peptide may be detected with aketoamine oxidase used in combination. For example, in the case of usinga ketoamine oxidase derived from Curvularia clavata YH 923 (FERMBP-10009), it is desirable that a glycated peptide cleaved from aglycated β-chain N-terminal of hemoglobin by a protease be FVH.

Although the ketoamine oxidase is not limited as long as it mayconstruct the above-described combination with a protease, desirable isa protease to cleave a glycated peptide rather than a glycated aminoacid from a β-chain N-terminal in terms of reactivity. Therefore, aglycated amino acid and/or a glycated peptide derived from a glycatedβ-chain N-terminal mentioned in (K1) to (K5) is desirably a glycatedpeptide derived from a glycated β-chain N-terminal. For example, aketoamine oxidase derived from Curvularia clavata YH 923 (FERM BP-10009)reacts with a glycated dipeptide derived from a glycated β-chainN-terminal (FVH) and does not substantially react with a glycateddipeptide derived from a glycated α-chain N-terminal (FVL), so that itmay be used as a ketoamine oxidase having the property (K3). Meanwhile,a mutant having reactivity to FZK of 5% or less compared to that to FVHmay be used as a ketoamine oxidase having the property (K1), andfructosyl amine oxidase derived from Fusarium oxysporum (manufactured byAsahi Kasei Pharma Corporation) may be used as a ketoamine oxidasehaving the property (K5).

<Reaction Condition>

The reaction condition of a protease to cleave a glycated amino acidand/or a glycated peptide derived from a glycated β-chain N-terminal ofglycated hemoglobin is not limited as long as it is sufficient forreaction proceeding. For example, desirable is generally a reactioncondition to obtain the property (P1) by regulating the pH, saltconcentration, added surfactant amount, added metallic ion amount,reaction temperature, added oxidant-reductant amount, or bufferconcentration to enhance the specificity of a protease having theproperty (P3) or (P2). In particular, for a neutral proteinase derivedfrom Bacillus sp. (manufactured by Toyobo Co., Ltd.), and proteasesderived from Bacillus sp. ASP-842 (FERM BP-08641), Aeromonas hydrophilaNBRC 3820, and Lysobacter enzymogenes YK-366 (FERM BP-10010), when aketoamine oxidase derived from Curvularia clavata YH 923 (FERM BP-10009)is used as a ketoamine oxidase to be combined, those proteasenecessarily become proteases having the property (P1) under a reactioncondition of pH 5.0 to 6.0, so that such reaction condition ispreferable.

The reaction condition of a ketoamine oxidase is not limited as long asit is sufficient for reaction proceeding. For example, desirable is areaction condition to obtain the reactivity to FZK of 30% or less byregulating the pH, salt concentration, added surfactant amount, addedmetallic ion amount, reaction temperature, added redox amount, or bufferconcentration. Particularly desirable is a reaction condition of pH 5.5to 6.5 because the specificity to FVH that is a glycated amino acidand/or glycated peptide derived from a β-chain N-terminal which iscleaved by a protease may be enhanced.

The correct determination of a glycated β-chain N-terminal of glycatedhemoglobin by the above-described method and reagent kit forspecifically determining a glycated β-chain N-terminal of glycatedhemoglobin using a protease and a ketoamine oxidase without a separationoperation may be confirmed by using a sample whose hemoglobin contentand glycation rate of a β-chain N-terminal of the hemoglobin arecorrectly shown. As the sample whose hemoglobin content and glycationrate of a 3-chain N-terminal of the hemoglobin are correctly shown, asample whose a determination value obtained by the method described inClinical Chemistry and Laboratory Medicine 40(1): 78-89 (2002) (IFCCvalue) is specified is desirably used. Alternatively, in a sample whosea determination value obtained by other method is specified, the IFCCvalue may be obtained by the conversion equation described in RinshoKensa 46(6), 729-734 (2002). Meanwhile, a sample of glycated hemoglobinmay be prepared from human blood by appropriately combining operationssuch as blood corpuscle separation, hemolysis, centrifugation, dialysis,ion-exchange chromatography, and affinity chromatography (ClinicalChemistry and Laboratory Medicine 36(5): 299-308 (1998)), or acommercially available sample may be purchased and used.

<Determining Object>

The determining object of the above-described method and reagent kit forspecifically determining a glycated β-chain N-terminal of glycatedhemoglobin using a protease and a ketoamine oxidase without a separationoperation is not limited as long as it includes glycated hemoglobin, andexamples thereof include a whole blood sample, hemolyzed blood cellsample, and purified hemoglobin sample.

Next, the present invention will be described by way of referenceexamples and examples.

Reference Example Production Method of Ketoamine Oxidase Derived fromCurvularia clavata YH 923 (FERM BP-10009)

Although Curvularia clavata YH 923 (FERM BP-10009) produces a ketoamineoxidase that reacts with FVH (hereinafter also referred to as FOD923),the amount of the produced ketoamine oxidase is small. Therefore, asshown below, FOD923 gene was expressed in Escherichia coli and purified,to thereby yield FOD923.

(1) Preparation of Curvularia clavata YH 923 (FERM BP-10009) ChromosomalDNA

100 ml of Sabouraud medium (glucose 4.0%, polypeptone 1.0%, pH 5.6) waspoured into a 500 ml-volume shaking flask and sterilized by anautoclave, and Curvularia clavata YH 923 (FERM BP-10009) was inoculatedthereto and cultured with shaking at 25° C. for 4 days, followed byfiltration of the culture medium using a No. 2 Filter Paper, to therebycollect the cells. The collected cells were frozen with liquid nitrogenand pulverized in a mortar to yield fine powder. Then, 15 ml of a bufferfor DNA extraction (5.0% SDS, 0.1 M NaCl, 50 mM Tris-HCl, pH 8.0) wasadded thereto, and the mixture was slowly shaken to dissolve the powder.Subsequently, the supernatant was collected by centrifugation (5,000rpm, 6 min, room temperature), and phenol/chloroform extraction andether extraction were performed three times and twice, respectively, toevaporate ethers remaining in the aqueous layer. Then, 1 ml of 3 Msodium acetate and 25 ml of ethanol were added thereto, and the mixturewas allowed to stand at −30° C. for 30 minutes. Thereafter, chromosomalDNA was collected by centrifugation (12,000 rpm, 10 min, 4° C.), washedwith 70% ethanol, and dissolved in 400 μl of TE. Subsequently, 10 μl(0.132 U) of RNase was added to the resultant DNA solution, and themixture was treated at 37° C. for 1 hour. Then, 5 μl (0.6 U) ofproteinase K was added thereto, and the mixture was treated at 50° C.for 1 hour. Thereafter, phenol/chloroform extraction (twice) andchloroform/isoamyl alcohol extraction (once) were performed, and ethanolprecipitation was performed to collect DNA. Subsequently, the collectedDNA was washed with a 70% ethanol solution, and ethanol was removed,followed by dissolution in 200 μl of TE, to thereby yield a chromosomalDNA solution.

(2) Amplification of FOD923 Gene Fragment

(a) Acquirement of FOD923 Gene Fragment

On the basis of known gene information of ketoamine oxidases, thefollowing primers P11 and P12 were designed.

P11 (SEQ ID NO: 2) AA (A/G) GC (C/T) AT (C/T) AACGC (C/T) AT (C/T) GGP12 (SEQ ID NO: 3) AC (C/G) ACGTGCTT (A/G) CC (A/G) ATGTT

35 cycles of PCR were performed using the chromosomal DNA obtained bythe above-described method as a template DNA and a primer P11 and aprimer P12 in combination. As a result, a DNA fragment having a size ofabout 800 bp was specifically amplified, and the amplified DNA fragment(sequence from the 1021st base to the 1790th base in SEQ ID NO: 1) wassequenced.

(b) FOD923 Gene Sequencing

A DNA fragment of the 5′ upstream region adjacent to the DNA fragmenthaving a size of about 800 bp obtained in (a) was amplified bycassette-ligation-medicated PCR using an LA-PCR in vitro Cloning kit(manufactured by Takara Bio Inc.) after digesting chromosomal DNA with arestriction enzyme XbaI. That is, XbaI cassette (manufactured by TakaraBio Inc.) was bound to a fragment obtained by a restriction enzymetreatment of the chromosomal DNA, and 35 cycles of PCR were performedusing the resultant DNA as a template DNA, a primer C1 (manufactured byTakara Bio Inc.), and the following primer P13. Then, 35 cycles of PCRwere further performed using a solution obtained by diluting thereaction solution 100-fold as a template, a primer C2 (manufactured byTakara Bio Inc.), and the following primer P14. As a result, a DNAfragment having a size of 1,200 bp was specifically amplified. Then, theamplified DNA fragment (sequence from the 1st base to the 1044th base inSEQ ID NO: 1) was sequenced.

A DNA fragment of the 3′ downstream region adjacent to the DNA fragmenthaving a size of about 800 bp obtained in (a) was amplified bycassette-ligation-medicated PCR using an LA-PCR in vitro Cloning kit(manufactured by Takara Bio Inc.) after digesting chromosomal DNA with arestriction enzyme SalI. That is, SalI cassette (manufactured by TakaraBio Inc.) was bound to a fragment obtained by a restriction enzymetreatment of the chromosomal DNA, and 35 cycles of PCR were performedusing the resultant-DNA as a template DNA, a primer C1 (manufactured byTakara Bio Inc.), and the following primer P15. Then, 35 cycles of PCRwere further performed using a solution obtained by diluting thereaction solution 100-fold as a template, a primer C2 (manufactured byTakara Bio Inc.), and the following primer P16. As a result, a DNAfragment having a size of 1,000 bp was specifically amplified. Then, theamplified DNA fragment (sequence from the 1775th base to the 2212th basein SEQ ID NO: 1) was sequenced.

The base sequences determined as above were ligated to determine a basesequence of FOD923 gene (SEQ ID NO: 1).

P13 GCCAAAAGAGGCTGCTTGAACGAT (SEQ ID NO: 38)(a complementary sequence to the base sequence from 1,083 to 1,106 ofSEQ ID NO: 1)

P14 GCATCCAGCACCACCAAAACAAAC  (SEQ ID NO: 39)(a complementary sequence to the base sequence from 1,062 to 1,086 ofSEQ ID NO: 1)

P15 TGGTGCCAGAACAACATGTACTGACC (SEQ ID NO: 40)(a base sequence from 1,713 to 1,738 of SEQ ID NO: 1)

P16 ACCTGGTTTGCCTAGGCACACA (SEQ ID NO: 41)(a base sequence from 1,736 to 1,757 of SEQ ID NO: 1)(3) Preparation of Escherichia coli Expressing Ketoamine Oxidase Derivedfrom Curvularia clavata YH 923 (FERM BP-10009)

In order to express FOD923 in Escherichia coli, three regions, i.e., thesequence from the 753rd base to the 807th base in SEQ ID NO: 1, sequencefrom the 1231st base to the 1279th base in SEQ ID NO: 1, and sequencefrom the 1696th base to the 1750th base in SEQ ID NO: 1 were estimatedas introns from the homology between a ketoamine oxidase derived fromPenicillium janthinellum (JP-A-11-46769) and a ketoamine oxidase derivedfrom Aspergillus nidulans.

The following operations were performed to remove introns and to createan expression plasmid in Escherichia coli.

First, there were synthesized: DNA fragments P22 to P27 having positiveand complementary sequences of a sequence including 40 to 50 bases so asto flank intron-existing regions to be removed; a DNA fragment P21obtained by adding a restriction sequence of a restriction enzyme NcoIonto the initiating codon ATG of FOD923 gene; and a DNA fragment P28having a complementary sequence obtained by adding a restriction enzymeSacI onto the site just behind the stop codon of FOD923 gene.

Subsequently, 4 kinds of PCR were performed using a chromosomal DNA ofCurvularia clavata YH 923 (FERM BP-10009) as a template and usingprimers P21 and P23, P22 and P25, P24 and P27, and P26 and P28respectively in combination, to thereby yield amplified DNA fragmentseach having a size of 327 bp, 480 bp, 471 bp, or 254 bp where intronregions had been removed. Then, the resultant four fragments were mixedand used as templates to perform PCR again using P21 and P28 as primers,to thereby yield a DNA fragment that has a size of about 1.4 kb and hasone FOD923 gene ligated to four fragments at three homologous regionswhere introns had been removed.

In order to produce FOD923 in Escherichia coli at a high level, therewas used a pyruvate oxidase promoter (JP-A-07-67390) derived fromAerococcus viridans that is a high expression promoter. Specificprocedures thereof will be described below.

1) In order to obtain a promoter region of a pyruvate oxidase gene froma plasmid pOXI3 including a pyruvate oxidase gene of Aerococcus viridansshown in JP-B-07-67390, pOXI3 was cleaved with DraI to separate apromoter region of a pyruvate oxidase gene that has a DNA sequencehaving a size of 202 bp described in SEQ ID NO: 6, and it was ligated toa fragment obtained by cleaving pUC13 with SalI and blunting it with T4DNA polymerase, to thereby yield a plasmid pKN19 where the direction ofan ampicillin-resistant gene in pUC13 and the direction of a pyruvateoxidase gene promoter are the same.

2) In order to perform more effective expression by a promoter, therewere synthesized: a DNA fragment P31 having a base sequence described inSEQ ID NO: 7, which was designed so as to provide a ribosome-bindingsequence region and multicloning site in the downstream of a pyruvateoxidase gene promoter of pKN19; and a DNA fragment P32 that is acomplementary sequence thereof and has a base sequence described in SEQID NO: 8. Then, annealing was performed, and the fragments were ligatedto pKN19 cleaved with XbaI and EcoRI, to thereby prepare a plasmidpPOS2.

3) A DNA fragment that has a size of about 1.4 kb and includes theabove-described FOD923 gene where introns had been removed was cleavedwith NcoI and SacI, and the resultant product was integrated in pPOS2cleaved with NcoI and SacI as well, to thereby prepare a plasmidpPOSFOD923 where FOD923 gene was integrated in the downstream of apyruvate oxidase gene promoter.

4) Escherichia coli W3110 was transformed with the resultant pPOSFOD923to create Escherichia coli FAOD923 that expresses FOD923.

P21 (SEQ ID NO: 4) CACACATCCTCGTCATTTCGCCATGGCGCCCTCAAGAGCAAAC P22CAAAGAGTATTTCCACAACACTGGAAGACTCGACTGTGCACATGGGGAAG AGG (SEQ ID NO: 42)(a base sequence from 725 to 752 and 808 to 832 of SEQ ID NO: 1) P23CCTCTTCCCCATGTGCACAGTCGAGTCTTCCAGTGTTGTGGAAATACTCT TTG (SEQ ID NO: 43)(a complementary sequence to the base sequence from 725 to 752 and 808to 832 of SEQ ID NO: 1) P24GACCTGGAAGATCAATGCGTTTCAAAAGCTTGGGTATATGCTCACATACA GCTTAC (SEQ ID NO:44) (a base sequence from 1,204 to 1,230 and 1,280 to 1,308 of SEQ IDNO: 1) P25 GTAAGCTGTATGTGAGCATATACCCAAGCTTTTGAAACGCATTGATCTTC CAGGTC(SEQ ID NO: 45) (a complementary sequence to the base sequence from1,204 to 1,230 and 1,280 to 1,308 of SEQ ID NO: 1) P26CTTTGTGCTGGCGACAGGGGACAGCGGGCACACATTCAAACTTTTGCCAA ATATC (SEQ ID NO: 46)(a base sequence from 1,669 to 1,695 and 1,751 to 1,778 of SEQ ID NO: 1)P27 GATATTTGGCAAAAGTTTGAATGTGTGCCCGCTGTCCCCTGTCGCCAGCA CAAAG (SEQ ID NO:47) (a complementary sequence to the base sequence from 1,669 to 1,695and 1,751 to 1,778 of SEQ ID NO: 1) P28 (SEQ ID NO: 5)CACGCTACAAGACGAGTTTCGAGCTCTATAACTTGGACTTGACAAC P31 (SEQ ID NO: 7)CTAGAGGAATAACACCATGGCCGTCGACGCTAGCATGCATGGATCCCGGG TACCGAGCTCG P32 (SEQID NO: 8) AATTCGAGCTCGGTACCCGGGATCCATGCAGCTAGCGTCGACGGCCATGG TGTTATTCCT(4) Production of Ketoamine Oxidase Derived from Curvularia clavataYH923 (FERM BP-10009)

The above-prepared Escherichia coli FAOD923 was inoculated in a 2.4 cmdiameter test tube that each contains 10 ml of a medium (pH 7.0)containing with 3% sorbitol, 1.5% peptone, 1.5% beer yeast extract, and50 μg/ml ampicillin and cultured with shaking at 28° C. for 12 hours, tothereby yield an inoculum. The inoculum was inoculated in a 30 L jarfermenter that contains 20 L of a medium (pH 7.0) containing 3%sorbitol, 1.5% peptone, 1.5% beer yeast extract, 0.1% antifoamer, and 50μg/ml ampicillin and cultured with stirring at 37° C. for 18 hours.

After completion of the culture, the cultured cells were collected andsuspended in 4 L of 10 mM Tris-HCl buffer (pH 7.5), and solubilized byultrasonic disintegration (212 KU). The solubilized solution wascentrifuged at 8,000 rpm for 30 minutes, and the supernatant wassubjected to ion-exchange chromatography using Q-Sepharose Big Beadsresin (2 L) (manufactured by Amersham). Meanwhile, elution was performedwith Tris-HCl buffer (pH 7.5) including 0 M, 0.1 M, 0.3 M, or 0.5 MNaCl. As a result, fractions eluted with buffers including 0.3 M and 0.5M NaCl were collected as active fractions (180 KU). The enzyme solutionswere concentrated to 750 ml by a module (manufactured by Amicon), andthe concentrated solution was dialyzed against 10 mM Tris-HCl buffer (pH7.5) overnight. The dialyzed solution was subjected to ion-exchangechromatography again using Q-Sepharose HP resin (500 ml) (manufacturedby Amersham). Meanwhile, elution was performed with Tris-HCl buffer (pH7.5) including 0 to 0.3 M NaCl by linear gradient, and fractions elutedwith buffers including 0.15 to 0.2 M NaCl (92 KU) were collected. Theenzyme solutions were concentrated to 500 ml, and the concentratedsolution was dialyzed against 10 mM Tris-HCl buffer (pH 7.5) overnight,followed by freeze-drying, to thereby yield purified FOD923. Thedetermination reagent and determination method for FOD923 activity willbe described below.

<Determination Reagent>

50 mM Tris-HCl buffer (pH 7.5)

1 mM FVH (manufactured by Peptide Institute, Inc.)

0.02% 4-aminoantipyrine (manufactured by Wako Pure Chemical Industries,Ltd.)

0.02% TOOS (manufactured by Dojindo Laboratories)

5 U/ml Peroxidase (manufactured by Sigma Corporation)

(TOOS: N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline)

<Determination Method>

1 ml of a determination reagent was poured into a test tube andpreheated at 37° C. for 5 minutes, and then 0.05 ml of an enzymesolution was added thereto, and the solution was allowed to react for 5minutes. After the reaction, 2 ml of 0.5% SDS was added to terminate thereaction, and the absorbance at a wavelength of 550 nm (Aa) wasdetermined. Meanwhile, as a blank, the same operations were performedusing a determination reagent including no FVH to determine theabsorbance (Ab). From the absorbance difference (Aa−Ab) between theabsorbance (Aa) and absorbance of the blank (Ab), the enzyme activitywas calculated.

One unit of the enzyme activity was defined as an enzyme amount forgeneration of 1 μmol of hydrogen peroxide at 37° C. for 1 minute.

Reference Example Production Method of Ketoamine Oxidase Derived fromNeocosmospora vasinfecta 474

Although Neocosmospora vasinfecta 474 produces a ketoamine oxidase thatreacts with FVH (hereinafter also referred to as FOD474), the amount ofthe produced ketoamine oxidase is small. Therefore, as shown below, theketoamine oxidase gene was expressed in Escherichia coli and purified,to thereby yield the ketoamine oxidase.

(1) Preparation of Chromosomal DNA of Neocosmospora vasinfecta 474

100 ml of Sabouraud medium (glucose 4.0%, polypeptone 1.0%, pH 5.6) waspoured into a 500 ml-volume Sakaguchi flask and sterilized by anautoclave, and Neocosmospora vasinfecta 474 was inoculated thereto andcultured with shaking at 25° C. for 4 days, followed by filtration ofthe culture medium using a No. 2 Filter Paper, to thereby collect thecells. The collected cells were frozen with liquid nitrogen andpulverized in a mortar to yield fine powder, and a chromosomal DNAsolution was obtained using a DNeasy Plant Maxi Kit (manufactured byQIAGEN).

(2) Cloning of Ketoamine Oxidase Gene

(a) Preparation of Radioactive DNA Probe

The ketoamine oxidase gene of Neocosmospora vasinfecta 474 was expectedto have the homology to FOD923 gene. Accordingly, the above-describedplasmid pPOSFOD923 including the FOD923 gene was cleaved withrestriction enzymes NcoI and SacI, and a DNA fragment that has a size ofabout 1.4 kb and includes a ketoamine oxidase gene was separated. Then,the DNA fragment was used with BcaBEST Labeling Kit (manufactured byTakara Bio Inc.) and [α-32P]dCTP to prepare a radioactive DNA probe.

(b) Assay of DNA Fragment Containing Ketoamine Oxidase Gene by SouthernHybridization

In order to create a gene library from the chromosomal DNA ofNeocosmospora vasinfecta 474 obtained by operations described in (1),operations to cleave the chromosome with various restriction enzymes andto assay the length of a DNA fragment containing a target gene wereperformed. First, the chromosomal DNA of Neocosmospora vasinfecta 474was cleaved with various restriction enzymes and subjected toelectrophoresis in a 1.5% agarose gel, and the DNA was transferred fromthe agarose gel to a nylon membrane (Biodyne A: manufactured by PALLcorporation). Next, the membrane was air-dried and immersed in ahybridization solution (0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 0.1%bovine serum albumin, 0.75 M sodium chloride, 75 mM sodium citrate, 50mM trisodium phosphate, 0.1% sodium dodecyl sulfate, 250 μg/ml salmonsperm DNA, and 50% formamide), and prehybridization was performed at 42°C. for 2 hours. After the prehybridization, the hybridization solutionwas exchanged for a new one, and the radioactive DNA probe created in(a) was added thereto, followed by a hybridization treatment overnightat 42° C. After the hybridization, the membrane was washed with awashing solution (75 mM sodium chloride, 7.5 mM sodium citrate, and 0.1%SDS) at 50° C. for 10 minutes and dried naturally. The dried membranewas placed on an X-ray film and exposed at −70° C. for 24 hours.

After the exposure, the film was developed, and there were observed thesizes of positive bands shown by the chromosomes cleaved with variousrestriction enzymes. As a result, it was found that a ketoamine oxidasegene is present on a DNA fragment having a size of about 8 kb which wasobtained by cleavage with SacI, and a gene library was to be createdusing the fragment of chromosomal DNA having a size of 8 kb which wasobtained by cleavage with SacI.

(c) Creation of Gene Library

The chromosomal DNA of Neocosmospora vasinfecta 474 obtained byoperations described in (1) was cleaved with a restriction enzyme SacI,and agarose electrophoresis was performed to separate a DNA fragmenthaving a size of about 8 kb. The resultant DNA fragment was ligated topUC119 being dephosphorylated with alkaline phosphatase using a DNALigation Kit (manufactured by Takara Bio Inc.) after being cleaved witha restriction enzyme SacI. Escherichia coli JM 109 competent cell(manufactured by Takara Bio Inc.) was transformed with the resultant andcultured on LB agar medium (manufactured by Becton, Dickinson andCompany) containing 50 μg/ml ampicillin, to thereby yield about 5,000ampicillin-resistant colonies, which were used as a gene library.

(d) Screening of Recombinant Escherichia coli Including DNA FragmentContaining Ketoamine Oxidase Gene by Colony Hybridization

The gene library obtained in (c) was replicated on a nylon membrane(Biodyne A: manufactured by PALL Corporation), and DNAs of the cellswere fixed thereon according to an appended manual of the membrane. TheDNA-fixed membrane was immersed in the hybridization solution shown in(b), and prehybridization was performed at 42° C. for 1 hour. After theprehybridization, the hybridization solution was exchanged for a newone, and the radioactive DNA probe created in (a) was added thereto,followed by a hybridization treatment overnight at 42° C. as well. Afterthe hybridization, the membrane was washed with the washing solutionshown in (b) at 50° C. for 10 minutes and dried naturally. The driedmembrane was placed on an X-ray film and exposed at −70° C. for 24hours. After the exposure, the film was developed, and 8 coloniesshowing positive signals were identified.

(e) Extraction of Recombinant Plasmid and Sequencing of KetoamineOxidase Gene

The colonies showing positive signals, which had been selected in (d),were inoculated in 1.5 ml of LB liquid medium (manufactured by Becton,Dickinson and Company) containing 50 μg/ml ampicillin and cultured withshaking at 37° C. for 16 hours, and plasmids were extracted. As aresult, the plasmids derived from 8 colonies were found to have the samechromosomal DNA fragment. For one of those plasmids, a region having thehomology to FOD923 gene was identified, and FOD474 gene was sequenced.SEQ ID NO: 9 shows the determined base sequence of FOD474 gene and aminoacid sequence encoded thereby.

(3) Construction of Escherichia coli Expressing Ketoamine OxidaseDerived from Neocosmospora vasinfecta 474

In order to express FOD474 in Escherichia coli, there were synthesized:a primer P41 (SEQ ID NO: 10) added with the recognition sequence of arestriction enzyme BspHI on an initiating codon ATG of FOD474 gene; anda primer P42 (SEQ ID NO: 11) having a complementary sequence added withthe recognition sequence of a restriction enzyme SacI on the site justbehind the stop codon of a ketoamine oxidase gene.

Subsequently, 25 cycles of PCR were performed using a chromosomal DNA ofNeocosmospora vasinfecta 474 as a template and P41 and P42 as primers,to thereby yield a DNA fragment having a size of about 1.4 kb andincluding FOD474 gene. The DNA fragment was cleaved with BspHI and SacI,and the resultant product was integrated into pPOS2 cleaved with NcoIand SacI to prepare a plasmid pPOSFOD474 where a ketoamine oxidase genewas integrated in the downstream of a pyruvate oxidase gene promoter.Meanwhile, a DNA fragment that has a size of about 1.4 kb and includes aFOD474 gene obtained by cleavage with XbaI and SacI, was integrated intoplasmid pUC19 to thereby create a plasmid p119-FOD474 (FERM BP-08642),and sequenced. As a result, it was confirmed that mutation due to PCRhad not occurred. Escherichia coli W3110 was transformed with theresultant pPOSFOD474 to construct Escherichia coli FAOD474 thatexpresses FOD474.

The plasmid p119-FOD474 (FERM BP-08642) has been deposited inInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi,Tsukuba, Ibaraki, Japan on Feb. 24, 2004.

(SEQ ID NO: 10) P41 TTTTTTCATGACCACCCCCCGCAAAGAAACCACCGTCCTC (SEQ ID NO:11) P42 TTTTTGAGCTCATCTTGACTCGCTGTCCTGATCGTGCTTC(4) Production of Ketoamine Oxidase Derived from Neocosmosporavasinfecta 474

Operations were performed using Escherichia coli FAOD474 in the same wayas the above-described FOD923. Meanwhile, the determination reagent forFOD474 activity and method were also the same as those of theabove-described FOD923.

Reference Example Substrate Specificities of Ketoamine Oxidase Derivedfrom Curvularia clavata YH923 (FERM BP-10009), Ketoamine Oxidase Derivedfrom Neocosmospora vasinfecta 474, and Ketoamine Oxidase Derived fromFusarium oxysporum (Manufactured by Asahi Kasei Pharma Corporation)

20 μl of an enzyme solution was added to 200 μl of a reaction solution(50 mM Tris-HCl buffer (pH 7.5), 0.1% Triton X-100, 0.03%4-aminoantipyrine, 0.02% TOOS, 5 U/ml peroxidase, and 2 mM substrate,the mixture was subjected to a reaction at 37° C. for 5 minutes, and 0.5ml of 0.5% SDS was then added. Absorbance at 555 nm (A1) was determinedand the same operations were performed using a reaction solutioncontaining no substrate to determine absorbance (Ab) to therebydetermining the reactivity from the difference (A1−Ab). Table 3 shows:the concentrations of used enzyme solutions of ketoamine oxidase derivedfrom FOD923, FOD474, and Fusarium oxysporum (manufactured by Asahi KaseiPharma Corporation: hereinafter also referred to as FOD2); usedsubstrates; absorbance differences (A1−Ab); and relative activities (%).

Reference Example Reaction for Releasing Fructosyl Valine from FVHL byAngiotensin-Converting Enzyme

An angiotensin-converting enzyme derived from porcine kidney(manufactured by Sigma Corporation: hereinafter referred to as ACEP) andan angiotensin-converting enzyme derived from rabbit lung (manufacturedby Sigma Corporation: hereinafter referred to as ACER) were dissolved inan enzyme-dissolving solution (100 mM HEPES (pH 8.3), 300 mM NaCl) so asto have a concentration of 20 U/ml, to thereby prepare an ACEP solutionand ACER solution. Subsequently, 20 μl of the enzyme-dissolvingsolution, ACEP solution, and ACER solution were separately added tothree tubes each containing 20 μl of 2 mM FVHL (manufactured by PeptideInstitute, Inc.) solution, and the mixture was allowed to react at 37°C. for 1 hour. Then, 200 μl of a coloring solution (50 mM Tris-HCl (pH7.5), 0.1% Triton X-100, 5 U/ml POD, 50 U/ml FOD2, and 0.02 mM DA-64)was added to each resultant, and the mixture was allowed to react at 37°C. for 5 minutes. Thereafter, 500 μl of 0.5% SDS was added to terminatethe coloring reaction, and the absorbance at 730 nm was determined. Thesame reaction was performed except that distilled water was used insteadof the 2 mM FVHL solution. Meanwhile, 200 μl of the coloring solutionwas added to 40 μl of an FV solution (0, 5, 10, 20, or 50 μM), anddetermination was performed in the same way as above. Then, acalibration curve was created, and the each amount of FV released fromFVHL by ACEP and ACER was determined. The calibration curve for FVrevealed in which absorbance differences at FV concentrations (μM) are0.019 at 5 μM, 0.033 at 10 μM, 0.065 at 20 μM, and 0.0154 at 50 μM,respectively. Meanwhile, the absorbance difference showing FV releasedby the ACEP reaction was found to be −0.006, while the absorbancedifference showing FV released by the ACER reaction was found to be0.000. Those results revealed that FV is hardly released from FVHL byACEP and ACER in a general reaction. Moreover, the absorbance differencein the case where the same determination was performed by adding thecoloring solution to 40 μl of a sample (25 μM FV, 10 U/ml ACER) wasfound to be 0.062, so that it was found that ACER hardly inhibits acoloring reaction in the coloring solution.

TABLE 3 (VHLT disclosed as SEQ ID NO:37) FOD923 FOD474 FOD2 FOD923Menzyme absorbance relative enzyme absorbance relative enzyme absorbancerelative enzyme absorbance relative conc. difference activity conc.difference activity conc. difference activity conc. difference activitySubstrate (U/ml) (A1 − Ab) (%) (U/ml) (A1 − Ab) (%) (U/ml) (A1 − Ab) (%)(U/ml) (A1 − Ab) (%) FZK 0.083 0.043 39 0.17 0.032 15 0.3 0.123 100 1.070.130 3.1 FV 0.025 0.217 556 0.05 0.289 445 1.5 0.109 18 0.016 0.290 464FVL 0.17 0.009 3.4 5 0.059 0.91 150 −0.001 0 1.07 0.050 1.2 FVH 0.050.078 100 0.17 0.221 100 150 −0.001 0 0.032 0.125 100 FVHL 50 0.067 0.0950 0.002 0 150 0.000 0 3.2 0.030 0.24 FVHLT 500 0.007 0.009 50 0.002 0150 0.001 0 32 0.028 0.022 β-glycated 500 0.000 0 50 0.002 0 150 0.001 032 0.043 0.034 pentapeptide α-glycated 50 0.008 0.01 50 0.003 0 150−0.001 0 32 0.003 0.002 pentapeptide

Example 1 Screening of Protease for Hemoglobin A1c Determination (pH7.5)

A screening method is shown in which a protease that cleaves a glycatedpeptide from N-terminal-glycated pentapeptide of a hemoglobin β-chainwithout substantially cleaving a glycated amino acid or glycated peptidefrom N-terminal-glycated pentapeptide of a hemoglobin α-chain. Each 10μl of a sample for screening was placed in 3 wells of a 96-well plate,respectively. Then, the first well was supplemented with 50 μl of anα-solution, the second well was supplemented with 50 μl of a β-solution,and the third well was supplemented with 50 μl of a control solution.After the plate was incubated at 37° C. for 60 minutes, 50 μl ofdistilled water and 50 μl of a developing solution were added, and thewhole was left at room temperature for 30 minutes. Then, the absorbanceat a measuring wavelength of 550 nm and a reference wavelength of 595 nmwere determined using a Microplate Reader (model 550, manufactured byBio-Rad), to select a sample having a difference in the absorbancebetween the β-solution-supplemented well and the controlsolution-supplemented well larger than a difference in the absorbancebetween the α-solution-supplemented well and the controlsolution-supplemented well. Thus the protease of object was screened.The samples to be used include: carboxypeptidase B (manufactured bySigma-Aldrich Corp.); carboxypeptidase W (manufactured by Wako PureChemical Industries, Ltd.); carboxypeptidase Y (manufactured by OrientalYeast Co., Ltd.); protease (type XXVII nagase: manufactured bySigma-Aldrich Corp.); protease (type VIII Subtilisin Carlsberg:manufactured by Sigma-Aldrich Corp.); protease (type XXIV Bacterial:manufactured by Sigma-Aldrich Corp.); Proteinase K (manufactured by WakoPure Chemical Industries, Ltd.); neutral proteinase (manufactured byToyobo Co., Ltd.); carboxypeptidase A (manufactured by Wako PureChemical Industries, Ltd.); thermolysin (manufactured by Wako PureChemical Industries, Ltd.); and distilled water. To confirm a degree ofcolor development caused by a developing solution, 10 μl of 1 mM FVH(manufactured by Peptide Institute Inc.) and 1 mM FVL (manufactured byPeptide Institute Inc.) were placed in a well severally, followed byadding 50 μl of the control solution. The whole was incubated at 37° C.for 60 minutes. Then, 50 μl of distilled water and 50 μl of thedeveloping solution were added and the whole was left at roomtemperature for 30 minutes, followed by the determination in a similarmanner as described above. The results are shown in Table 4 and Table 5.The results confirmed that carboxypeptidase B, carboxypeptidase W,neutral proteinase, and thermolysin were the proteases that cleave aglycated peptide from an N-terminal glycated pentapeptide of ahemoglobin β-chain without substantially cleaving a glycated amino acidor glycated peptide from N-terminal glycated pentapeptide of ahemoglobin α-chain at pH 7.5.

<α-Solution>

50 mM Tris-HCl (pH7.5)

0.1% Triton X-100

0.2 mM α-Glycated pentapeptide (manufactured by Peptide Institute Inc.)

<α-Solution>

50 mM Tris-HCl (pH7.5)

0.1% Triton X-100

0.2 mM β-Glycated pentapeptide (manufactured by Peptide Institute Inc.)

<Control Solution>

50 mM Tris-HCl (pH7.5)

0.1% Triton X-100

<Developing Solution>

100 mM Tris-HCl (pH7.5)

0.09% 4-Aminoantipyrine (manufactured by Wako Pure Chemical Industries,Ltd.)

0.06% TODB (manufactured by Dojindo Laboratories)

15 U/ml Peroxidase (manufactured by Sigma-Aldrich Corp.)

18.75 U/ml FOD923

(TODB: N,N-bis(4-sulfobutyl)-3-methylaniline)

TABLE 4 RAW DATA (pH 7.5) α-Solution β-Solution control solutioncarboxypeptidase B (100 U/ml) 0.036 0.127 0.028 carboxypeptidase W (520U/ml) 0.035 0.194 0.028 carboxypeptidase Y (1010 U/ml) 0.240 0.209 0.031protease type XXVII (810 U/ml) 0.021 0.031 0.016 protease type VIII(1100 U/ml) 0.023 0.020 0.012 protease type XXIV (1070 U/ml) 0.018 0.0330.010 proteinase K (520 U/ml) 0.031 0.077 0.017 neutral proteinase (4000U/ml) 0.029 0.196 0.026 distilled water 0.028 0.039 0.028 1 mMfructosyl-Val-His — — 0.188 1 mM fructosyl-Val-Leu — — 0.198

TABLE 5 RAW DATA (pH 7.5) α-Solution β-Solution control solutioncarboxypeptidase A (1000 U/ml) 0.022 0.145 0.018 thermolysin (75 kU/ml)0.053 0.249 0.055 distilled water 0.013 0.011 0.010 1 mMfructosyl-Val-His — — 0.169 1 mM fructosyl-Val-Leu — — 0.161

Example 2 Screening of Protease for Hemoglobin A1c Determination (pH3.5)

A screening method is shown in which a protease that cleaves a glycatedpeptide from N-terminal-glycated pentapeptide of a hemoglobin β-chainwithout substantially cleaving a glycated amino acid or glycated peptidefrom N-terminal-glycated pentapeptide of a hemoglobin α-chain. Each 10μl of a sample for screening was placed in 3 wells of a 96-well plate,respectively. Then, the first well was supplemented with 50 μl of anα-solution, the second well was supplemented with 50 μl of a β-solution,and the third well was supplemented with 50 μl of a control solution.After the plate was incubated at 37° C. for 60 minutes, 50 μl ofpH-regulating solution and 50 μl of a developing solution were added,and the whole was left at room temperature for 30 minutes. Then, theabsorbance at a measuring wavelength of 550 nm and a referencewavelength of 595 nm were determined using a Microplate Reader (model550, manufactured by Bio-Rad), to select a sample having a difference inthe absorbance between the β-solution-supplemented well and the controlsolution-supplemented well larger than a difference in the absorbancebetween the α-solution-supplemented well and the controlsolution-supplemented well. Thus the protease of object was screened.The samples to be used include: carboxypeptidase B (manufactured bySigma-Aldrich Corp.); carboxypeptidase W (manufactured by Wako PureChemical Industries, Ltd.); carboxypeptidase Y (manufactured by OrientalYeast Co., Ltd.); protease (type XXVII nagase: manufactured bySigma-Aldrich Corp.); protease (type VIII Subtilisin Carlsberg:manufactured by Sigma-Aldrich Corp.); protease (type XXIV Bacterial:manufactured by Sigma-Aldrich Corp.); Proteinase K (manufactured by WakoPure Chemical Industries, Ltd.); neutral proteinase (manufactured byToyobo Co., Ltd.); and distilled water. To confirm a degree of colordevelopment caused by a developing solution, 10 μl of 1 mM FVH(manufactured by Peptide Institute Inc.) and 1 mM FVL (manufactured byPeptide Institute Inc.) were placed in a well severally, followed byadding 50 μl of the control solution. The whole was incubated at 37° C.for 60 minutes. Then, 50 μl of a pH-regulating solution and 50 μl of thedeveloping solution were added and the whole was left at roomtemperature for 30 minutes, followed by the determination in a similarmanner as described above. The results are shown in Table 6. The resultsconfirmed that carboxypeptidase B was the protease that cleaves aglycated peptide from an N-terminal glycated pentapeptide of ahemoglobin β-chain without substantially cleaving a glycated amino acidor glycated peptide from N-terminal glycated pentapeptide of ahemoglobin α-chain at pH 3.5.

<α-Solution>

50 mM Acetate-sodium acetate (pH3.5)

0.1% Triton X-100

0.2 mM α-Glycated pentapeptide (manufactured by Peptide Institute Inc.)

<β-Solution>

50 mM Acetate-sodium acetate (pH3.5)

0.1% Triton X-100

0.2 mM β-Glycated pentapeptide (manufactured by Peptide Institute Inc.)

<Control Solution>

50 mM Acetate-sodium acetate (pH3.5)

0.1% Triton X-100

<pH-Regulating Solution>

100 mM CAPS—NaOH (pH 11.0)

(CAPS: 3-cyclohexylaminopropane sulfonic acid: manufactured by DojindoLaboratories)

<Developing Solution>

100 mM Tris-HCl (pH7.5)

0.09% 4-aminoantipyrine (manufactured by Wako Pure Chemical Industries,Ltd.)

0.06% TODB (manufactured by Dojindo Laboratories)

15 U/ml Peroxidase (manufactured by Sigma-Aldrich Corp.)

18.75 U/ml FOD923

(TODB: N,N-bis(4-sulfobutyl)-3-methylaniline)

TABLE 6 RAW DATA (pH 3.5) α-Solution β-Solution control solutioncarboxypeptidase B (100 U/ml) 0.063 0.147 0.060 carboxypeptidase W (520U/ml) 0.211 0.184 0.032 carboxypeptidase Y (1010 U/ml) 0.195 0.206 0.034protease type XXVII (810 U/ml) 0.025 0.021 0.019 protease type VIII(1100 U/ml) 0.019 0.018 0.016 protease type XXIV (1070 U/ml) 0.013 0.0120.012 proteinase K (520 U/ml) 0.024 0.026 0.023 neutral proteinase (4000U/ml) 0.036 0.040 0.035 distilled water 0.038 0.040 0.038 1 mMfructosyl-Val-His — — 0.192 1 mM fructosyl-Val-Leu — — 0.180

Example 3 Screening of Protease for Hemoglobin A1c Determination Derivedfrom Microorganisms

Various bacteria were cultured with shaking in Difco Lactobacilli MRSBroth (manufactured by Becton, Dickinson and Company), a MM medium (2.5%mannose, 2.5% beer yeast extract, pH7.0), and a YPG medium (2% glucose,1% polypeptone, 2% yeast extract, 0.1% potassium dihydrogen phosphate,0.05% magnesium sulfate heptahydrate, pH 7.0) at a temperature of from28 to 30° C. for 1 to 7 days. Then, cells were removed by centrifugationfrom the culture solutions to obtain culture supernatants. A sample wasprepared by diluting the obtained culture supernatants 10-fold with a 10mM potassium phosphate buffer (pH7.5), and the determination wasperformed in a similar manner as Example 1. Table 7 shows the resultsfrom the bacteria cultured in MRS Broth, Table 8 shows the results fromthe bacterium cultured in the MM medium, and Table 9 shows the resultsfrom the bacterium cultured in the YPG medium (note that a referencewavelength was not determined in Table 9).

TABLE 7 RAW DATA (pH 7.5) α-Solution β-Solution control solutionBacillus sp. (FERM BP-08641) 0.063 0.229 0.058 distilled water 0.0400.033 0.049 1 mM fructosyl-Val-His — — 0.218 1 mM fructosyl-Val-Leu — —0.238

TABLE 8 RAW DATA (pH 7.5) α-Solution β-Solution control solutionBacillus subtilis NBRC3037 0.062 0.198 0.066 distilled water 0.018 0.0210.015 1 mM fructosyl-Val-His — — 0.172 1 mM fructosyl-Val-Leu — — 0.181

TABLE 9 RAW DATA (pH 7.5) α-Solution β-Solution control solutionLysobacter enzymogenes 0.170 0.696 0.168 YK-366 (FERM BP-10010)Aeromonas hydrophila 0.156 0.510 0.152 NBRC3820

Example 4 Method of Purifying Protease Derived from Bacillus sp. ASP842(FERM BP-08641) and Physicochemical Property Thereof

Bacillus sp. ASP842 (FERM BP-08641) were cultured with shaking in four500 ml-Erlenmeyer flasks which was supplemented with 150 ml of DifcoLactobacilli MRS Broth (manufactured by Becton, Dickinson and Company)at 28° C. for 3 days. Then, cells were removed from the culturesolutions by centrifugation to obtain a culture supernatant (77 mU/ml,560 ml). The culture supernatant was added with 210 g of ammoniumsulfate and 8.4 g of perlite (manufactured by Toko Perlite Industry Co.,Ltd.) and the whole was stirred. Then, precipitated proteases werecollected by filtration with Filter Paper No. 5A (manufactured byToyo-Roshi Kaisha, Ltd.) having a diameter of 90 mm. Subsequently, theprecipitants was suspended in a 10 mM potassium phosphate buffer (pH7.5), followed by filtration using Filter Paper 5A (manufactured byToyo-Roshi Kaisha, Ltd.), thereby obtaining a filtrate having a proteaseactivity (215 mU/ml, 90 ml). The filtrate was subjected to dialysisagainst 5 L of a 10 mM potassium phosphate buffer (pH7.5). The dialyzedprotease solution was adsorbed to DEAE-Sepharose FF (manufactured byAmersham) in a column (26φ×94 mm) equilibrated with a 10 mM potassiumphosphate buffer (pH 7.5), and a fraction having protease activity (217mU/ml, 54 ml) was obtained by elution with NaCl gradient of 0 M to 0.5M. After ammonium sulfate was added to be the concentration of 1 M inthe fraction, the fraction was adsorbed to Phenyl-Sepharose CL-4B(manufactured by Amersham) in a column (15φ×150 mm) equilibrated with 1M ammonium sulfate and a 10 mM potassium phosphate buffer (pH 7.5).Then, a fraction having protease activity (237 mU/ml, 18 ml) was elutedwith a gradient of 1M ammonium sulfate, 0% ethylene glycol to 0Mammonium sulfate, 20% ethylene glycol, and concentrated with AmiconUltra 10000 MWCO (manufactured by Millipore), thereby to obtain apurified protease (13.9 U/ml, 0.42 ml). The reagent and method fordetermining the activity of the protease of the invention are describedbelow.

<Determination Reagent>

50 mM Tris-HCl (pH7.5)

2 mM Calcium chloride

0.1% Triton X-100

0.03% 4-Aminoantipyrine

0.02% TOOS

5 U/ml Peroxidase

5 U/ml FOD923

0.25 mM Substrate

<Determination Method>

0.2 ml of a determination reagent was placed in a test tube andpreheated at 37° C. for 5 minutes. Then, 0.02 ml of an enzyme solutionwas added thereto and a reaction was carried out for 10 minutes. Afterthe reaction, 0.5 ml of 0.5% SDS was added thereto to terminate thereaction. Then, absorbance at the wavelength 555 nm (Aa) was determined.In addition, distilled water was added as a blank instead of the enzymesolution to determine absorbance (Ab) by performing a similar operation.An enzymatic activity was obtained from a difference between theabsorbance (Aa) and the absorbance of a blank (Ab) (Aa−Ab). One unit ofthe enzyme activity was defined as an amount of an enzyme that allowsgenerating 1 μmol of hydrogen peroxide per minute at 37° C. Note that,β-glycated pentapeptide was used as a substrate.

<Physicochemical Properties>

(1) Substrate Specificity

Determination was carried out according to <Determination Method> inExample 4, except that the concentration of FOD923 was changed to 50U/ml and 0.25 mM β-glycated pentapeptide, 0.25 mM α-glycatedpentapeptide, 0.03 mM FVH, 0.03 mM FVL, and 0.03 mM FV were used assubstrate. In addition, similar determination in which FOD923 is changedto FOD2 was performed. Furthermore, similar determination was performedusing neutral protease (manufactured by Toyobo Co., Ltd.) forcomparison. Table 10 shows difference in the obtained absorbances(Aa−Ab). The results confirmed that the protease of the inventioncleaved FVH from β-glycated pentapeptide without cleaving a glycatedamino acid or glycated peptide from α-glycated pentapeptide, in the samemanner as neutral protease (manufactured by Toyobo Co., Ltd.) cleaves.

TABLE 10 FOD 923 FOD 2 neutral protease neutral protease RAW DATA DWproteinase ASP842 DW proteinase ASP842 β-glycated 0.044 0.417 0.2580.030 0.028 0.029 pentapeptide α-glycated 0.036 0.036 0.034 0.025 0.0200.025 pentapeptide FVH 0.163 0.159 0.150 0.024 0.044 0.024 FVL 0.1720.173 0.165 0.024 0.024 0.022 FV 0.152 0.161 0.162 0.216 0.209 0.217 DW0.030 0.031 0.030 0.026 0.022 0.024(2) Optimum pH

FIG. 2 shows the results of determination according to the determinationmethod described in Example 4, except that 50 mM Tris-HCl (pH 8.0, 8.5),50 mM PIPES (pH 6.0, 6.5, 7.0) and 50 mM citrate-sodium citrate (pH 5.5,6.0, 6.5) were used instead of 50 mM Tris-HCl (pH 7.5) respectively and0.25 mM substrate was changed to 0.1 mM β-glycated pentapeptide. Theresults confirmed that pH of about 6.0 is optimum.

(3) pH Stability

The enzyme was treated with 50 mM Tris-HCl (pH 7.5, 8.0, 8.5) and 50 mMPIPES (pH 6.0, 6.5, 7.0) at 50° C. for 20 minutes and a residualactivity thereof was determined. FIG. 3 shows the results.

(4) Optimum Temperature

20 μl of a protease solution were added to 100 μl of a reaction solution(50 mM Tris-HCl (pH 7.5), 2 mM calcium chloride, 0.1% Triton X-100, 2.5mM β-pentapeptide). A reaction was carried out at 30, 37, 42, 50, 55,60, and 65° C. (10 minutes for each temperature) followed by thetermination of the reaction by adding 5 μL of 0.5 M EDTA. Then, 95 μl ofa developing solution (0.03% 4-aminoantipyrine, 0.02% TOOS, 50 U/mlperoxidase, 10.5 U/ml FOD923) was added thereto and a reaction wascarried out at 37° C. for 5 minutes. Subsequently, 95 μl of 0.5% SDS wasadded thereto and absorbance at 555 nm was determined, thereby todetermine the optimum temperature. FIG. 4 shows the results.

(5) Thermal Stability

The protease was placed in 50 mM Tris-HCl (pH 7.5) and treated at 4, 37,50, 60, and 70° C. (10 minutes for each temperature), followed bydetermining residual activities. FIG. 5 shows the results.

(6) Molecular Weight

35 kDa (SDS-PAGE)

32,721 Da (MALDI-TOF MASS analysis)

Example 5 Culture Method and Purification Method for Protease Derivedfrom Aeromonas hydrophila NBRC 3820 Strain and Enzymological PropertiesThereof

<Culture Method>

Each 100 ml of a YPG medium (2.0% glucose, 1.0% polypeptone, 2.0% yeastextract, 0.1% KH2PO4, 0.05% MgSO4.7H2O, pH 7.0) was placed in ten 500ml-Sakaguchi flasks and sterilized, followed by inoculating Aeromonashydrophila NBRC 3820. The whole was cultured with shaking at 30° C. for2 days.

<Purification Method>

The obtained culture solution was subjected to centrifugation. Ammoniumsulfate was added to the obtained culture supernatant to be 40%saturated and the centrifuged supernatant was provided to a column ofButyl Toyopearl 650M (30φ×150 mm, manufactured by Tosoh Corp.)equilibrated with 0.1 M Tris-HCl buffer (pH 7.3) in which 40% saturatedammonium sulfate was added. Washing was performed with 0.1 M Tris-HClbuffer (pH 7.3) containing ammonium sulfate added to be 10% saturated,and elution was performed with 0.1 M Tris-HCl buffer (pH 7.3). After anactive fraction of the eluate was dialyzed against a 50 mM Tris-HClbuffer (pH 7.3), the active fraction was provided to a column ofDEAE-Sepharose Fast Flow (30φ×150 mm, manufactured by Amersham)equilibrated with the same buffer and was subjected to elution with NaClhaving a gradient of 0 to 0.5 M. Ammonium sulfate was added to an activefraction of the eluate to be 40% saturated. The whole was provided to acolumn of Butyl-Toyopearl 650 M (18φ×150 mm, manufactured by TosohCorp.) equilibrated with 0.1 M Tris-HCl buffer (pH 7.3) in which 40%saturated ammonium sulfate was added, and was subjected to elution withsaturated ammonium sulfate having a linear gradient of 40% to 0%. Activefractions were collected and dialyzed against distilled water therebyobtaining a purified protease. Table 11 shows procedures of thepurification.

The activity of the enzyme of the invention was determined as follows.0.45 ml of a 100 mM Tris-HCl buffer (pH 7.5) in which β-glycatedpentapeptide was dissolved to be 0.5 mM was placed in a cell having anoptical path length of 1 cm. The whole was preheated at 37° C. for 5minutes followed by adding 0.05 ml of an enzyme solution, and a reactionwas carried out for 10 minutes. After the reaction, 0.5 ml of adetermination reagent (a 100 mM Tris-HCl buffer (pH 7.5) containing0.04% TOOS, 0.06% 4-aminoantipyrine, peroxidase 10 U, and ketoamineoxidase 10 U derived from Curvularia clavata YH923) was added thereto.After color was developed for 2 minutes, 2 ml of 0.5% SDS was added toterminate the reaction, then absorbance at the wavelength 550 nm (Aa)was determined. In addition, absorbance (Ab) was determined byperforming a similar operation using various buffer solutions containingno substrates as blank. An enzymatic activity was obtained from adifference between the absorbance (Aa) and the absorbance of a blank(Ab) (Aa−Ab).

TABLE 11 Purification of protease derived from Aeromonas hydrophilaNBRC3820 total specific protein total activity activity recoverypurification steps (A₂₈₀) (U) (U/A₂₈₀) (%) 40-80% ammonium 32,500 4550.01 100.0 sulfate precipitation Butyl-Toyopearl 650M 31.5 302 9.60 66.5DEAE-sepharose FF 13.4 132 9.82 29.0 Butyl-Toyopearl 650M 11.4 112 9.8524.7<Physicochemical Properties>(1) Actions

Table 12 shows actions of the protease derived from Aeromonas hydrophilaNBRC 3820 of the present invention on α-glycated pentapeptide andβ-glycated pentapeptide. Concentrations of respective substrates duringthe reaction were set to be 0.25 mM. The protease derived from Aeromonashydrophila NBRC 3820 (1.0 U) was added thereto and a reaction wascarried out at 30° C. for 5 to 60 minutes. Then, the reaction solutionwas brought into a protein sequencer (manufactured by Shimadzu Corp.)and an amino acid sequence at N-terminal of the peptide produced by theaction of the protease was determined.

As the result, the protease derived from Aeromonas hydrophila NBRC 3820did not act on α-glycated pentapeptide, while it cleaved a peptide bondbetween histidine and leucine in β-glycated pentapeptide and producedFVH and leucyl-threonyl-proline (LTP).

(2) Optimum pH

0.45 ml of various buffers having pH of 3.0 to 11.0 (a 100 mM acetatebuffer with pH 3-5, a 100 mM citrate buffer with pH 5-7, a 100 mMTris-HCl buffer with pH 7-9, and a 100 mM borate buffer with pH 9-11) inwhich β-glycated pentapeptide was dissolved to be 0.5 mM respectivelywere placed in cells having an optical path length of 1 cm. The wholewas preheated at 37° C. for 5 minutes followed by adding 0.05 ml of anenzyme solution, and a reaction was carried out for 10 minutes. Afterthe reaction, 0.5 ml of a determination reagent (a 100 mM Tris-HClbuffer (pH 7.5) containing 0.04% TOOS, 0.06% 4-aminoantipyrine,peroxidase 10 U, and ketoamine oxidase 10 U derived from Curvulariaclavata YH923) was added thereto. After color was developed for 2minutes, 2 ml of 0.5% SDS was added to terminate the reaction, thenabsorbance at the wavelength 550 nm (Aa) was determined. In addition,absorbance (Ab) was determined by performing a similar operation usingvarious buffer solutions containing no substrates as blank. An enzymaticactivity was obtained from a difference between the absorbance (Aa) andthe absorbance of a blank (Ab) (Aa−Ab). FIG. 6 shows the results.

(3) pH Stability

The enzyme solution was treated with 10 mM various buffers at 4° C. for24 hours and residual activities thereof were determined according tothe activity determination method described in <Purification Method>.FIG. 7 shows the results.

(4) Optimum Temperature

0.45 ml of a 100 mM Tris-HCl buffer (pH 7.5) in which β-glycatedpentapeptide was dissolved to be 0.5 mM was placed in a cell having anoptical path length of 1 cm. The whole was preheated at from 15 to 70°C. for 5 minutes followed by adding 0.05 ml of an enzyme solution, and areaction was carried out for 10 minutes. After the reaction, the wholewas cooled on ice. Then, 0.5 ml of a determination reagent (a 100 μMTris-HCl buffer (pH 7.5) containing 0.04% TOOS, 0.06% 4-aminoantipyrine,peroxidase 10 U, and ketoamine oxidase 10 U derived from Curvulariaclavata YH923) was added thereto. After color was developed for 2minutes, 2 ml of a 0.5% SDS solution was added to terminate thereaction, then absorbance at the wavelength 550 nm (Aa) was determined.In addition, absorbance (Ab) was determined by performing a similaroperation using various buffer solutions containing no FVH as blank. Anenzymatic activity was obtained from a difference between the absorbance(Aa) and the absorbance of a blank (Ab) (Aa−Ab). FIG. 8 shows theresults for an optimum temperature obtained by changing temperature from15 to 70°.

(5) Thermal Stability

The enzyme solution was treated with a 100 mM Tris-HCl buffer (pH 7.5)for 30 minutes for each temperature and residual activities thereof weredetermined according to the activity determination method for the enzymedescribed above. FIG. 9 shows the results.

(6) Molecular Weight

The molecular weight of the enzyme of the invention was obtained by gelfiltration using YMC-Pack Diol-200G (6.0φ×300 mm, manufactured by YMC).Bovine serum albumin, ovalbumin, and a soybean trypsin inhibitor (allmanufactured by Sigma Corp.) were used as standard proteins. As aresult, the molecular weight was about 33,000.

SDS-PAGE (sodium dodesyl sulfate-polyacrylamide gel electrophoresis)using 10% gel of Laemmli's method resulted in a molecular weight ofabout 33,000. Note that, SDS-PAGE Standard Low (manufactured by Bio-Rad)was used as a standard protein.

The above results revealed that the protease of the invention derivedfrom Aeromonas hydrophila NBRC 3820 is a monomer.

(7) Amino Acid Sequence of N-Terminal Portion

According to the above method, a purified enzyme obtained from a culturesolution of Aeromonas hydrophila NBRC 3820 was dissolved in distilledwater and subjected to analysis using an N-terminal sequencer (PPSQ-21,manufactured by Shimadzu Corp.). The obtained sequence was as shown bySEQ ID NO: 12. Homology search was conducted for the obtained amino acidsequence of the N-terminal portion with known proteases, resulting thatthe obtained amino acid sequence of the N-terminal portion has 98%homology to an elastase derived from Aeromonas hydrophila.

(SEQ ID NO: 12) Val Asp Ala Thr Gly Pro Gly Gly Asn Val Lys Thr Gly LysTyr Phe Tyr Gly(8) Partial Amino Acid Sequence

Segments of the enzyme were obtained to determine its sequence,according to “Method of purifying minor proteins for microsequence, p48-52, 1992, Yodosha Co., Ltd.”, using 60 g of a freeze-dried product ofthe purified enzyme obtained from the culture solution of Aeromonashydrophila NBRC3820 according to the above method. That is, 50 μl of 45mM dithiothreitol was added and the whole was treated at 50° C. for 15minutes, followed by adding 5 μl of 100 mM iodoacetoamide and being leftfor 15 minutes. Subsequently, 140 μl of distilled water and 1.0 μg ofendoproteinase Lys-C (manufactured by Roche Diagnostics) were addedthereto and the whole was incubated overnight at 37° C. The segmentedenzymes were separated by HPLC thereby obtaining respective enzymefragments. The respective fragments were subjected to analysis using theN-terminal sequencer (PPSQ-21, manufactured by Shimadzu Corp.). Thefollowing SEQ ID NOS: 13 and 14 show the obtained sequences. The twoobtained partial amino acid sequences were completely matched with theamino acid sequence of the elastase derived from of Aeromonashydrophila, respectively.

(SEQ ID NO: 13) Leu Asp Val Ala Ala His Glu Val Ser His (SEQ ID NO: 14)Phe Gly Asp Gly Ala Thr

Table 12 shows the above-described properties of the protease derivedfrom Aeromonas hydrophila NBRC 3820.

TABLE 12 Aeromonas hydrophilaNBRC3820 Molecular weight (Da) SDS-PAGE33,000 Optimal reaction pH pH 6.5-7.0  (37 degree C., 10 min) pHstability (5 degree C., 24 h) pH 6.0-11.0 Optimal reaction temperature 50 degree C. (pH 7.5, 10 min) Thermal stability ~60 degree C. (pH 7.5,10 min)

  100

N.D. F-VLSPA (α-chain) N.D. (SEQ ID NO: 50) Elastin degradation +N-terminal amino acid sequence VNATGPGGNVKTGKYFYG (SEQ ID NO: 48)Reaction pattern

Example 6 Culture Method and Purification Method for Protease Derivedfrom Lysobacter enzymogenes YK-366 (FERM BP-10010) Strain andEnzymological Properties thereof

<Purification Method>

Active fractions were collected by the same method as the productionmethod and purification method described in Example 5 and dialyzedagainst distilled water to thereby obtaining a purified protease. Table13 shows process of the purification. An activity determination methodto be performed was the same as the method in Example 5.

TABLE 13 Purification of protease derived from Lysobacter enzymogenesYK-366 total total protein activity specific activity recoverypurification steps (A₂₈₀) (U) (U/A₂₈₀) (%) 40-80% ammonium 39,500 5240.01 100.0 sulfate precipitation Butyl-Toyopearl 650M 36.8 261 7.09 49.8DEAE-sepharose FF 5.6 49 8.75 9.4 Butyl-Toyopearl 650M 5.1 41 8.04 7.8<Physicochemical Properties>(1) Actions

Table 14 shows actions of the protease derived from Lysobacterenzymogenes YK-366 of the present invention on α-glycated pentapeptideand β-glycated pentapeptide. The action of the protease derived fromLysobacter enzymogenes YK-366 on α-glycated pentapeptide and β-glycatedpentapeptide was investigated according to the method described in (1)of Example 5. As a result, the protease derived from Lysobacterenzymogenes YK-366 did not act on α-glycated pentapeptide, while itcleaved a peptide bond between histidine and leucine in β-glycatedpentapeptide, and produced FVH and LTP.

(2) Optimum pH

FIG. 10 shows the results obtained by investigating the optimum pH ofthe protease derived from Lysobacter enzymogenes YK-366 according to themethod described in (2) of Example 5.

(3) pH Stability

FIG. 11 shows the results obtained by investigating the pH stability ofthe protease derived from Lysobacter enzymogenes YK-366 according to themethod described in (3) of Example 5.

(4) Optimum Temperature

FIG. 12 shows the results obtained by investigating the optimumtemperature of the protease derived from Lysobacter enzymogenes YK-366according to the method described in (4) of Example 5.

(5) Thermal Stability

FIG. 13 shows the results obtained by investigating the thermalstability of the protease derived from Lysobacter enzymogenes YK-366according to the method described in (5) of Example 5.

(6) Molecular Weight

As the results of obtaining the molecular weight by the gel filtrationand SDS-PAGE according to the method described in (6) of Example 5, themolecular weight was about 35,000 respectively.

The above result revealed that the protease of the present inventionderived from Lysobacter enzymogenes YK-366 is a monomer.

(7) Amino Acid Sequence of N-Terminal Portion

According to the above method, the purified enzyme obtained from theculture solution of Lysobacter enzymogenes YK-366 was dissolved indistilled water and subjected to analysis using the N-terminal sequencer(PPSQ-21, manufactured by Shimadzu Corp.). The obtained sequence was asshown by SEQ ID NO: 15.

(SEQ ID NO: 15) Ala Leu Val Gly Thr Gly Pro Gly Gly Asn Gln Lys Thr GlyGln Tyr Glu Tyr Gly Thr(8) Partial Amino Acid Sequence

Segments of the enzyme were obtained to determine sequence thereof,according to a conventional method (“Method of purifying minor proteinsfor microsequence, p 48-52, 1992, Yodosha Co., Ltd.”), using 60 g of afreeze-dried product of the purified enzyme obtained from the culturesolution of Lysobacter enzymogenes YK-366 according to the above method.That is, 50 μl of 45 mM dithiothreitol was added and the whole wastreated at 50° C. for 15 minutes, followed by adding 5 μl of 100 mMiodoacetoamide and being left for 15 minutes. Subsequently, 140 μl ofdistilled water and 1.0 μg of endoproteinase Lys-C (manufactured byRoche Diagnostics) were added thereto and the whole was incubatedovernight at 37° C. The segmented enzymes were separated by HPLC therebyobtaining respective enzyme fragments. The respective fragments weresubjected to analysis using an N-terminal sequencer (PPSQ-21,manufactured by Shimadzu Corp.). The following SEQ ID NOS: 16 and 17show the obtained sequences.

Tyr Ser Xaa Asn Tyr Glu Asn Ala (SEQ ID NO: 16) Phe Gly Asp Gly Ala Thr(SEQ ID NO: 17)

Table 14 shows the above-described properties of the protease derivedfrom Lysobacter enzymogenes NBRC 3820.

TABLE 14 Lysobacter enzymogenes YK-366 Molecular weight (Da) SDS-PAGE35,000 Optimal reaction pH pH 6.0-6.5  (37 degree C., 10 min) pHstability (5 degree C., 24 h) pH 5.0-11.0 Optimal reaction temperature 50 degree C. (pH 7.5, 10 min) Thermal stability ~60 degree C. (pH 7.5,10 min)

  100

N.D. F-VLSPA (α-chain) N.D. (SEQ ID NO: 50) Elastin degradation +N-terminal amino acid sequence ALVGTGPGGNQKTGQYEYGT (SEQ ID NO: 49)Reaction pattern

Example 7 Acquisition of Mutant Ketoamine Oxidase having DecreasedActivity on FZK by Gene Modification

<Construction of Mutant Library of Ketoamine Oxidase Derived fromCurvularia clavata YH923>

Construction of a mutant library of FOD923 was created from error-pronePCR that utilizes misreading of DNA polymerase. The error-prone PCRemployed pPOSFOD923 described in the above reference example as atemplate, was performed using a P51 primer and a P52 primer (given inthe following SEQ ID NOS: 18 and 19) and Ready-To-Go PCR beads(manufactured by Amersham) such that magnesium chloride was added to bea final concentration of 2.5 mM. PCR was conducted under the cycleconditions of: 94° C.×40 seconds, 55° C.×30 seconds, 72° C.×1 minute;and 25 cycles.

(SEQ ID NO: 18) P51 ACACCATGGCGCCCTCAAGAGCAAACACT (SEQ ID NO: 19) P52TTCGAGCTCTATAACTTGGACTTGACAACATCGTC

An amplified PCR product was purified using a GFX PCR DNA and Gel BandPurification-kit (manufactured by Amersham).

0.2 μg of the purified PCR product was digested by restriction enzymesNcoI and SacI and collected by ethanol precipitation. Meanwhile, 0.5 μgof pPOSFOD923 was also digested by the same restriction enzymes, and aDNA fragment of 2.9 kbp containing no ketoamine oxidase gene wasseparated by agarose-gel electrophoresis and collected using a GFX PCRDNA and Gel Band Purification-kit. Both DNA fragments described abovewere subjected to a reaction using a Ligation High kit (manufactured byToyobo Co., Ltd.) at 16° C. for 30 minutes to ligate each other. Byusing the ligated DNA fragment, a competent cell of Escherichia coliJM109 was transformed. The transformant was spread on an LB agar mediumcontaining 50 μg/ml of ampicillin and static cultured at 37° C. untilcolonies grew to about 1-2 mm.

<Screening of Mutant Ketoamine Oxidase Having Decreased Activity on FZK>

100 μl of a preliminary sterilized expression medium (a mediumcontaining 3% of sorbitol, 1.5% of polypeptone, 1.5% of yeast extract,and 50 μg/ml of ampicillin and adjusted to pH 7.0) was added to a96-well microplate. Grown transformants were inoculated thereto andcultured overnight at 30° C. After the termination of the culture, each5 μl of the culture solution was dispensed into two wells of a 96-wellplate and 50 μl of a determination reagent (a 50 mM Tris-HCl buffer (pH7.5) containing 1 mM FVH or FZK, 0.02% TOOS, 0.02% 4-aminoantipyrine and10 U/ml peroxidase) was added to each well, and a reaction was carriedout at 30° C. for 10-60 minutes. 150 μl of 0.5% SDS was added thereto toterminate the reaction and absorbance was determined at 550 nm using aMicroreader (model 450, manufactured by Bio-Rad). From the constructedmutant library, a recombinant which produces ketoamine oxidase that acton FVH but has a decreased activity on FZK was selected. As a result ofthe screening, 923-F1 and 923-F2 were obtained as favorable mutantstrains. Ketoamine oxidase genes harbored by those mutant strains weresequenced and resulted that mutations were introduced in the basesindicated by Table 15.

TABLE 15 corresponding amino acid restriction gene mutation mutationenzyme used FZK/FVH FOD923 — — — 1.1 923-F1 185 g→a 62 R→H — 0.24 923-F2172 a→g 58 I→V — 0.025 185 g→a 62 R→H 988 t→c 330 F→L  923-I58V 172 a→g58 I→V XbaI 0.12 923-F330L 988 t→c 330 F→L  NcoI 0.95 Bst1107I 923-Fro2172 a→g 58 I→V NcoI 0.025 185 g→a 62 R→H Bst1107I Note: The describedbase numbers are counted from the position 471, which is considered asthe first position, in the base sequence according to SEQ ID NO: 1,except intron sites.<Confirmation of Substrate Specificity of Mutant Ketoamine Oxidase>

Colonies from the selected strain was inoculated to 5 ml of an LB medium(containing 50 μg/ml of ampicillin) and cultured with shaking at 30° C.for 20 hours. Cells were collected from the culture solution after thetermination of the culture by centrifugation, were suspended in 5 ml ofa 0.1 M Tris-HCl buffer (pH 7.5), and were solubilized by ultrasonicbreaking. The solubilized solution was subjected to centrifugation(8,000 rpm, 10 minutes) and the obtained supernatant was used as a crudeenzyme solution of ketoamine oxidase. Specificity (a specific activityof the activity on FZK to the activity on FVH; indicated as FZK/FVHhereinafter) was obtained using those crude enzyme solutions and FVH andFZK as substrates according to the activity determination methoddescribed in <Screening of Mutant Ketoamine Oxidase having DecreasedAction on FZK>. As a result, FZK/FVH ratios of 923-F1 and 923-F2 were0.24 and 0.025 respectively as shown in Table 15, revealed that theactivities on FZK of the mutant enzymes have largely decreased.

<Identification of Effective Mutational Position>

To identify an effective mutational position that affected onimprovement of the above-described substrate specificity, mutantketoamine oxidase having introduced the point mutations indicated inTable 15 was created by the following method.

1) Mutant enzymes 923-F330L and 923-Fro2 were obtained from Escherichiacoli transformed by using a plasmid obtained by: treating a gene of awild type or mutation-type enzyme 923-F2 with the restriction enzymesindicated by Table 15, based on the restriction enzyme map of theplasmid pPOSFOD923 shown in FIG. 14; cleaving a fragment containing themutational position of interest; and inserting the fragment into theother plasmid treated with the same restriction enzymes.

2) A mutant enzyme 923-I58V was obtained from Escherichia colitransformed by using a plasmid obtained by: performing PCR with mutantprimers P53 and P54 (indicated by SEQ ID NOS: 20 and 21) to amplify aregion containing the mutation of interest; treating the obtainedfragment with the restriction enzymes shown in Table 15; and insertingthe fragment into pPOSFOD923 cleaved by the same restriction enzymes.Table 15 shows the result of evaluation of those mutant enzymes, by thedetermination method described in <Screening of Mutant Ketoamine Oxidasehaving Decreased Action on FZK>. It revealed that a substitution ofisoleucine of amino acid no. 58 (I58) by valine or a substitution ofarginine of amino acid no. 62 (R62) by histidine is effective todecrease the activity on FZK.

P53

CGACTCTAGAGGAATAACACCATGGCGCCCTC (SEQ ID NO: 20, including a XbaI sitelocating at downstream of the multicloning site of pPOS2)

P54

GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATGACCT TAT (SEQ ID NO:21, a complementary sequence to the sequence from 637 to 694 of SEQ IDNO: 1 containing a mutation such that the amino acid 581 is substitutedby V, in FOD923)

<Test for Point Mutation of Effective Mutant Amino Acid Residue>

The isoleucine of the highly effective amino acid no. 58 (I58) wassubstituted by another amino acid to search for an effective pointmutation-type ketoamine oxidase. P53 (SEQ ID NO: 20) and primers(P55-64) having the sequence indicated in (Table 15-2) were used tocreate a mutant strain by the method shown in 2) of <Identification ofEffective Mutational Position>. Results obtained by determining anactivity of the mutant enzyme of the activity on FZK to the activity onFVK by the method described in <Screening of Mutant Ketoamine Oxidasehaving Decreased Action on FZK> was shown in (Table 15-2).

The results revealed that substitutions of isoleucine of the amino acidno. 58 (I58) by threonine, asparagine, cysteine, serine, and alaninewere also effective for decreasing the activity on FZK, other than byvaline.

TABLE 15-2 codon of used primer (complementary chain) amino acid AminoSEQ at 58^(th) Acid Primer ID FZK/ Strain position Mutation No. SequenceNO FVH FOD923 atc — — — — 0.95 923-F2 gtc 158V, — — 0.025 R62H, F330L923-I58V gtc I58V P54GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATGACCTTAT 21 0.12923-I58F ttt I58F P55GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATAAACTTAT 22 6.4923-I58L ctg I58L P56GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATCAGCTTAT 23 1.6923-I58M atg I58M P57GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATCATCTTAT 24 2.3923-I58T acc I58T P58GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATGGTCTTAT 25 0.12923-I58A gcg I58A P59GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATCGCCTTAT 26 0.30923-I58Y tat I58Y P60GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATATACTTAT 27 54923-I58N aac I58N P61GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATGTTCTTAT 28 0.16923-I58C tgc I58C P62GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATGCACTTAT 29 0.16923-I58S agc I58S P63GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATGCTCTTAT 30 0.19923-I58G ggc I58G P64GCTTCTAGACTCAATTGGAGATCGACCTTGTTCCGCAAGCGGATACCCATGCCCTTAT 31 0.73<Production of Mutant Ketoamine Oxidase>

Mutant ketoamine oxidase was purified by the same method as described inthe reference example and produced.

Specificity of the mutant fructosyl aminoxidase of the present inventionto various substrates are as shown in Table 16. Note that, concentrationof each substrate was set to be 1 mM and other reaction conditions wereset according to the activity determination method described in<Screening of Mutant Ketoamine Oxidase having Decreased Action on FZK>.The results indicated that 923-F2 (hereinafter, also called as FOD923M)has the highest reactivity to FV and has also high reactivity to FVH,while it has reactivities of 2.5% and 0.5% to FZK and FVL respectivelyand has little reactivities compared to that of FVH, indicating that theenzyme of the invention is an enzyme that does not substantially act onFZK and FVL.

TABLE 16 Substrate FOD 923 923-F1 923-F2 FVH 100 100 100 FZK 95 24 2.5FV 630 680 220 FVL 2.6 — 0.5<Mutant of Ketoamine Oxidase Derived from Neocosmospora vasinfecta 474>

Ketoamine oxidase derived from Neocosmospora vasinfecta 474 (FOD474) hashigh homology to ketoamine oxidases containing FOD923 and its amino acidsequence at an N-terminal region containing 158 of FOD923 is highlyconserved as shown in FIG. 1. Using pPOSFOD474, FOD474 mutant strain474-F1 in which 158 of FOD474 is substituted by a different amino acid(threonine) was acquired. Table 17 shows specificity of FOD474 and474-F1 to various substrates. For the substrate specificity to FVH alsowas improved in FOD474, it indicated that the amino acid region no. 58is a site that strongly affect on the specificity to FVH even in a caseof a ketoamine oxidase derived from other microorganism species.

TABLE 17 Substrate FOD 474 474-F1 FVH 100 100 FZK 40 20 FV 540 380

Example 8 pH Dependency of Activity of Ketoamine Oxidase FOD923, FOD474,and FOD923M on FVH and FZK

20 μl of an enzyme solution was added to 200 μl of a reaction solution(50 mM buffer, 0.1% Triton X-100, 0.03% 4-aminoantipyrine, 0.02% TOOS, 5U/ml peroxidase, 2 mM substrate). The whole was incubated at 37° C. for5 minutes and then 500 μl of 0.5% SDS was added thereto followed bydetermining absorbance at 555 nm. Tris-HCl (pH 7.5, 8.0, 8.5), PIPES (pH6.0, 6.5, 7.0), and Bistris-HCl (pH 5.0, 5.5, 6.0, 6.5) were used as thebuffers for determination. Also, absorbance was determined using areaction solution containing no substrates. Differences in theabsorbance when FVH and FZK each were used as a substrate andconcentrations of the enzyme in the enzyme solution to be used aredescribed in Table 18. Table 19 shows a specific activity (%)represented by (FZK/FVH) when FVH and FZK each were used as a substrate.The results confirmed that the specificity to FVH significantlyincreases when pH is around 6.

Example 9 Substrate Specificity of Ketoamine Oxidase FOD923M

Determination was performed by the same method described in <ReferenceExample: Substrate Specificity of Ketoamine Oxidase derived fromCurvularia clavata YH923 (FERM BP-10009), Ketoamine Oxidase derived fromNeocosmospora vasinfecta 474, and Ketoamine Oxidase derived fromFusarium oxysporum (manufactured by Asahi Kasei Pharma)>. Table 3 showsthe results.

TABLE 18 Enzyme conc. Bistris PIPES Tris (U/ml) substrate 5.0 5.5 6.06.5 6.0 6.5 7.0 7.5 8.0 8.5 FOD923 0.1 FVH −0.002 0.020 0.048 0.0630.057 0.073 0.070 0.066 0.042 0.025 0.1 FZK −0.001 0.005 0.011 0.0150.013 0.022 0.033 0.030 0.041 0.049 FOD474 0.167 FVH 0.023 0.084 0.1870.273 0.265 0.303 0.243 0.163 0.104 0.063 0.833 FZK 0.012 0.022 0.0420.063 0.066 0.093 0.132 0.162 0.200 0.210 FOD923M 0.04 FVH 0.005 0.0570.168 0.189 0.192 0.181 0.075 0.062 0.017 0.009 0.64 FZK −0.001 0.0120.030 0.045 0.033 0.038 0.045 0.036 0.049 0.060

TABLE 19 Bistris PIPES Tris 5.0 5.5 6.0 6.5 6.0 6.5 7.0 7.5 8.0 8.5FOD923 — 25.0 22.9 23.8 17.8 30.1 47.1 45.5 97.6 196.0 FOD474 10.4 5.24.9 4.6 5.0 6.1 10.9 19.9 38.5 66.7 FOD923M — 1.3 1.1 1.5 1.1 1.3 3.83.6 18.0 41.7

Example 10 Determination of Hemoglobin A1c

To create a calibration curve, 324 μl of R1 (−protease) and 90 μl of R2are added to 36 μl of a hemoglobin A1c standard solution having FVHadded thereto, in a cell having an optical path length of 1 cm. Afterthe whole is incubated at 37° C. for 200 seconds, 10 μl of R3 is addedand the whole is incubated at 37° C. for 100 seconds. Subsequently, 10μl of R4 is added thereto and the whole is incubated at 37° C. for 100seconds. During the process, absorbency at 730 nm 180 seconds after R2was added (A1s), absorbency at 730 nm 90 seconds after R3 was added(A2s), and absorbency at 730 nm 90 seconds after R4 was added (A3s) aredetermined. Similarly, the same operation is performed with a hemoglobinA1c standard solution having no FVH added thereto, and absorbency at 730nm 180 seconds after R2 was added (A1sb), absorbency at 730 nm 90seconds after R3 was added (A2sb), and absorbency at 730 nm 90 secondsafter R4 was added (A3sb) are also determined. The calibration curveshown in FIG. 15 was created from the relationship between thedifference of the absorbance ΔAs=(A3s−A2s)−(A3sb−A2sb) and the FVHconcentrations.

Next, 324 μl of R1 (+protease) was added to 36 μl of each low or highlevel hemoglobin A1c standard solution. The whole was incubated at 37°C. for 60 minutes followed by adding 90 μL of R2 thereto. The whole wasincubated at 37° C. for 200 seconds in a cell having an optical pathlength of 1 cm followed by adding 10 μl of R3 thereto and incubating at37° C. for 100 seconds. Subsequently, 10 L of R4 was added thereto andthe whole was incubated at 37° C. for 100 seconds. During the process,absorbency at 730 nm 180 seconds after R2 was added (A1), absorbency at730 nm 90 seconds after R3 was added (A2), and absorbency at 730 nm 90seconds after R4 was added (A3) are determined. In addition, the sameoperation as described above using the same sample except R1 (−protease)was used instead of R1 (+protease) was carried out, and absorbency at730 nm 180 seconds after R2 was added (A1b), absorbency at 730 nm 90seconds after R3 was added (A2b), and absorbency at 730 nm 90 secondsafter R4 was added (A3b) are also determined. Amount of a glycatedβ-chain N-terminal of hemoglobin in each low or high level hemoglobinA1c standard solution can be obtained from the relationship between thedifference of the absorbance ΔA=(A3−A2)−(A3b−A2b) and the FVHconcentrations. As shown in Table 20, the theoretical values agree wellwith the determined values. Note that, the difference of the absorbanceΔAε=(A2−A1)−(A2b−A1b) is considered to be proportional to the amount ofglycated ε-amino groups of lysine residues in the glycated hemoglobin.

<R1 (+Protease)>

20 mM Tris-HCl (pH 7.5)

0.1% Triton X-100

150 mM Sodium chloride

2 mM Calcium chloride

2.1 kU/ml neutral proteinase derived from Bacillus sp. (manufactured byToyobo Co., Ltd.)

<R1 (−Protease)>

20 mM TrisvHCl (pH 7.5)

0.1% Triton X-100

150 mM Sodium chloride

2 mM Calcium chloride

<R2>

20 mM Tris-HCl (pH 7.5)

6.35 mM WST3 (manufactured by Dojindo Laboratories)

0.08 mM DA-64 (manufactured by Wako Pure Chemical Industries, Ltd.)

25 U/ml Peroxidase (manufactured by Sigma-Aldrich Corp.)

(WST-3:2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium)

(DA-64: N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine)

<R3>

500 U/ml FOD2

<R4>

500 U/ml FOD923

<Low Level Hemoglobin A1c Standard Solution>

To prepare, an available freeze-dried product of calibrator forhemoglobin determination (Determiner Control for HbA1c: manufactured byKyowa Medex Co., Ltd.) having a low value (represented hemoglobin A1cvalue (JDS value): 5.6%) was dissolved in distilled water to be 4 mg/ml.

By a conversion using the equation (JDS value)=0.9259 (IFCCvalue)+1.6697, described in “Rinsho Kensa, 46 (6) 729-734, 2002”, thehemoglobin A1c value (IFCC value) is converted to be 4.2%. Consequently,when hemoglobin is a tetramer consisting of two α-chains and twoβ-chains and has a molecular weight of 64,550, a theoretical value forthe concentration of the glycated β-chain N-terminals in this standardsolution will be 0.0052 mM.

<High level Hemoglobin A1c Standard Solution>

To prepare, an available freeze-dried product of calibrator forhemoglobin determination (Determiner Control for HbA1c: manufactured byKyowa Medex Co., Ltd.) having a high value (represented hemoglobin A1cvalue (JDS value): 10.2%) was dissolved in distilled water to be 4mg/ml.

By a conversion using the equation (JDS value)=0.9259 (IFCCvalue)+1.6697, described in “Rinsho Kensa, 46 (6) 729-734, 2002”, thehemoglobin A1c value (IFCC value) is converted to be 9.2%. Consequently,when hemoglobin is a tetramer consisting of two α-chains and twoβ-chains and has a molecular weight of 64,550, a theoretical value forthe concentration of the glycated β-chain N-terminals in this standardsolution will be 0.0114 mM.

<Hemoglobin A1c Standard Solution Having FVH Added Thereto>

To prepare, FVH (manufactured by Peptide Institute Inc.) was added tothe above low level hemoglobin A1c standard solution to be 0.030 mM and0.015 mM.

<Hemoglobin A1c Standard Solution Having No FVH Added Thereto>

The above low level hemoglobin A1c standard solution was used.

Example 11 Relationship between Reaction Time for Protease andDetermined Value of Hemoglobin A1c

Reaction times for protease in the low and high hemoglobin A1c standardsolutions used in Example 10 varied from 30 minutes to 360 minutes. FIG.16 shows the obtained determined values of hemoglobin A1c. It indicatesthat the agreement of the theoretical value and the determined value inExample 10 is not accidentally obtained by limiting the reaction timefor protease to be 60 minutes. It also indicates that the determinedvalues are almost stable with time when the reaction time for proteasevaries from 30 minutes to 360 minutes and therefore an actual amount ofglycated β-chain N-terminals in glycated hemoglobin is determined by thepresent determination method.

Example 12 1) Evaluation of Effect of pH in Degradation Reaction ofGlycated Hemoglobin by Neutral Proteinase Derived from Bacillus sp.(Manufactured by Toyobo Co., Ltd.)

1-a) Case where the Amount of FVH is Determined Using FOD923 afterGlycated Lysine and Peptides Containing the Glycated Lysine wereDigested by FOD2, After a Protease Reaction

30.4 μl of 20 mM WST3 and 36 μl of a glycated hemoglobin sample wereadded to 324 μl of a R1 reaction solution (20 mM buffer, surfactant,sodium chloride, 2 mM calcium chloride, 1.2 kU/ml neutral proteinase(manufactured by Toyobo Co., Ltd.)). After 60 seconds, 44.6 μl of a R2reaction solution (100 mM buffer, 50 U/ml peroxidase, 0.16 mMDA-64) wasadded thereto, and 5 μl of 0.5 M EDTA and 15 μl of a pH-regulatingsolution were added thereto after further 300 seconds. After further 50seconds, 10 μl of 1,500 U/ml of FOD2 was added thereto, 5 μl of 500 U/mlof FOD923 was added thereto after further 250 seconds, and the whole wassubjected to a reaction for 150 seconds. All reactions were carried outat 37° C. in a cell of an absorptiometer. The absorbance at 730 nm wasmonitored to obtain a difference (A1) between the absorbance at 240seconds after FOD2 was added and the absorbance at 140 seconds afterFOD923 was added. FIG. 17 shows a scheme of the determination.

A similar operation was carried out in a case where FVH waspreliminarily added to the R1 reaction solution to be 3.33 μM, to obtaina difference of absorbances (A2). Also, the same operation except that 5μl of 0.5 M EDTA and 15 μl of a pH-regulating solution was added beforeglycated hemoglobin was added, not after a R2 reaction solution wasadded, was carried out thereby obtaining a difference of absorbances ina blank reaction (Ab).

The FVH that was cleaved from glycated hemoglobin by protease can becalculated from an equation “determined value (μM)=(A1−Ab)/(A2−A1)×30”with those differences of absorbances. Note that, EDTA was added toterminate or inhibit the reaction of the protease. The pH-regulatingsolution was added to adjust pH at a reaction of ketoamine oxidase FOD2and FOD923 to be about 7.5. Combinations of the buffer, surfactant,concentration of sodium chloride, and a pH-regulating solution to beused in the R1 and R2 reaction solutions and determinations thereof wereshown in Table 21.

1-b) Case where the Amount of FVH is Determined Using FOD923 Such thatGlycated Lysine and Peptide Containing the Glycated Lysine are NotDigested by FOD2, after a Protease Reaction

30.4 μl of 20 mM WST3 was added to 324 μl of a R1 reaction solution (20mM buffer, surfactant, sodium chloride, 2 mM calcium chloride, 1.2 kU/mlneutral proteinase (manufactured by Toyobo Co., Ltd.)) and 36 μl of aglycated hemoglobin sample was added thereto. After 60 seconds, 44.6 μlof a R2 reaction solution (100 mM buffer, 50 U/ml peroxidase, 0.16 mMDA-64) was added thereto, and 5 μl of 0.5 M EDTA and 15 μl of apH-regulating solution were added thereto after further 300 seconds.After further 50 seconds, 5 μl of 500 U/ml of FOD923 was added thereto,and the whole was subjected to a reaction for 150 seconds. All reactionswere carried out at 37° C. in a cell of an absorptiometer. Theabsorbance at 730 nm was monitored to obtain a difference (A1) betweenthe absorbance at 40 seconds after the 0.5 M EDTA and a pH-regulatingsolution were added and the absorbance at 140 seconds after FOD923 wasadded. FIG. 18 shows a scheme of the determination.

A similar operation was carried out in a case where FVH waspreliminarily added to the R1 reaction solution to be 3.33 μM, to obtaina difference of absorbances (A2). Also, the same operation except that 5μl of 0.5 M EDTA and 15 μl of a pH-regulating solution was added beforeglycated hemoglobin was added, not after R2 reaction solution was added,was carried out thereby obtaining a difference of absorbances in a blankreaction (Ab). The FVH that was cleaved from glycated hemoglobin byprotease can be calculated from an equation “determined value(μM)=(A1−Ab)/(A2−A1)×30” with those differences of absorbances. Notethat, EDTA was added to terminate or inhibit the reaction of theprotease. The pH-regulating solution was added to adjust pH at areaction of ketoamine oxidase FOD2 and FOD923 to be about 7.5.Combinations of the buffer, surfactant, concentration of sodiumchloride, and a pH-regulating solution to be used in the R1 and R2reaction solutions and determinations thereof were shown in Table 21.

TABLE 20 measured value theoretical value ΔA [mM] [mM] Low levelhemoglobin A1c 0.0172 0.0045 0.0052 standard solution High levelhemoglobin A1c 0.0430 0.0107 0.0114 standard solution

(2) Evaluation of Effect of pH in Degradation Reaction of GlycatedHemoglobin by Protease Derived from Bacillus sp. ASP842

2-a) Case where the Amount of FVH is Determined Using FOD923 afterGlycated Lysine and Peptides Containing the Glycated Lysine are Digestedby FOD2, after a Protease Reaction

For the R1 reaction solution in 1-a) of Example 12, a protease derivedfrom Bacillus sp. ASP842 was added thereto to be 0.85 U/ml instead of1.2 kU/ml neutral proteinase (manufactured by Toyobo Co., Ltd.). Anoperation similar to 1-a) was carried out using combinations of thebuffer, surfactant, concentration of sodium chloride, and apH-regulating solution to be used in the R1 and R2 reaction solutions asshown in Table 22. Table 22 also shows the determination.

2-b) Case where the Amount of FVH is Determined Using FOD923, Such thatGlycated Lysine and Peptides Containing the Glycated Lysine are NotDigested by FOD2, after a Protease Reaction

For the R1 reaction solution in 1-b) of Example 12, a protease derivedfrom Bacillus sp. ASP842 was added thereto to be 0.85 U/ml instead of1.2 kU/ml neutral proteinase (manufactured by Toyobo Co., Ltd.). Anoperation similar to 1-b) was carried out using combinations of thebuffer, surfactant, concentration of sodium chloride, and apH-regulating solution to be used in the R1 and R2 reaction solutions asshown in Table 22. Table 22 also shows the determination.

(3) Evaluation of Effect of pH in Degradation Reaction of GlycatedHemoglobin by Protease Derived from Lysobacter enzymogenes YK-366

30.4 μl of 20 mM WST3, 36 μl of glycated hemoglobin sample, and 5 μl ofdistilled water were added to a R1 reaction solution (20 mM buffer,surfactant, sodium chloride, 2 mM calcium chloride, 0.43 U/ml proteasederived from Lysobacter enzymogenes YK-366). After 60 seconds, 44.6 μlof a R2 reaction solution (100 mM buffer, 50 U/ml peroxidase, 0.16 mMDA-64) was added thereto, followed by adding 15 μl of a pH-regulatingsolution after further 300 seconds. After 50 seconds, 5 μl of 500 U/mlof FOD 923 was added thereto and a reaction was brought about for 150seconds. All reactions were carried out at 37° C. in a cell of anabsorptiometer. The absorbance at 730 nm was preliminarily monitored toobtain a difference (A1) between the absorbance at 40 seconds after thepH-regulating solution was added and the absorbance at 140 seconds afterFOD923 was added. FIG. 19 shows a scheme of the determination.

TABLE 21 pH  5.0  5.5  6.0  6.5  7.0  7.5 Buffer MES Bistris BistrisBistris Tris Tris Surfactant 0.4% 0.3% 0.2% 0.1% 0.1% 0.2% Triton X-100Triton X-100 Triton X-100 Briji 35 Triton X-100 Triton X-100 NaCl Conc.150 mM 150 mM 150 mM 225 mM 150 mM 150 mM pH-regulating solution 1M Tris1M Tris 1M Tris 1M Tris Distilled water Distilled water (pH 9.0) (pH9.0) (pH 8.5) (pH 8.5) 1-a) measured value [,,M] 30.7 30.3 29.0 30.529.2 30.0 1-b) measured value [,,M] 29.7 30.7 28.8 32.7 36.1 34.6

TABLE 22 pH  5.0  5.5  6.0  7.0  7.5 Buffer Bistris Bistris Bistris TrisTris Surfactant 0.4% 0.3% 0.2% 0.2% 0.2% Triton X-100 Triton X-100Triton X-100 Triton X-100 Triton X-100 NaCl Conc. 150 mM 150 mM 150 mM150 mM 150 mM pH-regulating solution 1M Tris 1M Tris 1M Tris Distilledwater Distilled water (pH 9.0) (pH 9.0) (pH 8.5) 2-a) measured value[,,M] 29.5 28.2 29.2 29.3 31.4 2-b) measured value [,,M] 29.2 31.6 27.735.3 34.2

TABLE 23 pH  5.5  6.0  6.5  7.0  7.5 Buffer Bistris Bistris Bistris TrisTris Surfactant 0.3% 0.2% 0.2% 0.1% 0.2% Triton X-100 Triton X-100Triton X-100 Triton X-100 Triton X-100 NaCl Conc. 150 mM 150 mM 150 mM150 mM 150 mM pH-regulating solution 1M Tris 1M Tris 1M Tris Distilledwater Distilled water (pH 9.0) (pH 8.5) (pH 8.5) measured value [,,M]27.5 26.7 26.8 36.6 35.3

A similar operation was carried out in a case where FVH waspreliminarily added to the R1 reaction solution to be 3.33 μM, to obtaina difference of absorbances (A2). Also, a similar operation using aprotease inactivated by preliminarily treating it at 95° C. for 10minutes was carried out thereby obtaining a difference of absorbances ina blank reaction (Ab).

The FVH that was cleaved from glycated hemoglobin by a protease can becalculated from an equation “determined value (μM)=(A1−Ab)/(A2−A1)×30”with those differences of absorbances.

An operation similar to one in 1-b) of Example 12 was carried out usingcombinations of the buffer, surfactant, concentration of sodiumchloride, and a pH-regulating solution to be used in the R1 and R2reaction solutions as shown in Table 23. Determinations thereof wereshown in Table 23.

A glycated hemoglobin sample such that 5.9 mg/ml thereof is 16.7% interms of IFCC value (a theoretical value for concentration of glycatedβ-chain N-terminal is to be 30.5 μM) was used. The results shown inTable 21 to Table 23 indicate that: the protease does not cleave apeptide containing glycated lysine which can be acted with FOD923 outfrom glycated hemoglobin, when pH in a protease reaction to be 5.0-6.0.Therefore, the results indicated that the amount of FVH was specificallyand precisely determined (the amount of glycated β-chain N-terminals ofthe glycated hemoglobin) without digested by FOD2, i.e. the amount ofhemoglobin A1c was precisely determined.

Example 13 Determination of FVH in Degradation Product of GlycatedHemoglobin by Protease with Mutant Ketoamine Oxidase FOD923 Having HighSpecificity

1-a) Case where the Amount of FVH is Determined Using FOD 923M afterGlycated Lysine and Peptides Containing the Glycated Lysine are Digestedby FOD2, after a Protease Reaction

An operation similar to one in 1-a) of Example 12 was carried out,except Tris-HCl (pH 7.5) as the buffer of a R1 reaction solution, 0.1%Triton X-100 as the surfactant, and concentration of sodium chloride of150 mM were used, and 5 μl of 32 U/ml of FOD923M instead of 5 μl of 500U/ml of FOD923 was added. From determination, the determined value forFVH was 28.6 μM.

1-b) Case where the Amount of FVH is Determined Using FOD923M Such thatGlycated Lysine and Peptides Containing the Glycated Lysine are NotDigested by FOD2, after a Protease Reaction

An operation similar to one in 1-b) of Example 12 was carried out,except Tris-HCl (pH 7.5) as the buffer of a R1 reaction solution, 0.1%Triton X-100 as the surfactant, and concentration of sodium chloride of150 mM were used, and 5 μl of 32 U/ml of FOD923M instead of 5 μL of 500U/ml of FOD923 was added. From determination, the determined value forFVH was 30.0 μM.

From the above, the use of ketoamine oxidase having high specificityallows determination for the amount of FVH (the amount of glycatedβ-chain N-terminals of glycated hemoglobin) specifically without beingdigested by FOD2 under pH 7.5 (i.e. a condition in which the proteasecleaves glycated lysine and peptides containing the glycated lysine fromglycated hemoglobin), i.e. it allows precise determination for theamount of hemoglobin A1c.

INDUSTRIAL APPLICABILITY

A method of specifically determining glycated β-chain N-terminals ofglycated hemoglobin using enzymes without operating to separate and akit involving determination reagents can be provided.

[Reference to Deposited Biological Materials]

(1)

i. Name and address of depository institution at which the biologicalmaterial of interest is deposited.

International Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology

Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan (post code:305-8566)

ii. Date when the biological material was deposited to the depositoryinstitution of i.

Feb. 12, 2003 (original deposit date)

Apr. 12, 2004 (date of transfer to the deposition under Budapest Treatyfrom the original deposition)

iii. Accession number for the deposition assigned by the depositoryinstitution of i.

FERM BP-10009

(2)

i. Name and address of depository institution to which the biologicalmaterial of interest is deposited

International Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology

Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan (post code:305-8566)

ii. Date when the biological material was deposited to the depositoryinstitution of i.

Feb. 24, 2004 (original deposit date)

iii. Accession number for the deposition assigned by the depositoryinstitution of i.

FERM BP-08641

(3)

i. Name and address of depository institution to which the biologicalmaterial of interest is deposited

International Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology

Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan (post code:305-8566)

ii. Date when the biological material was deposited to the depositoryinstitution of i.

Jan. 30, 2004 (original deposit date)

Apr. 12, 2004 (date of transfer to the deposition under Budapest Treatyfrom the original deposition)

iii. Accession number for the deposition assigned by the depositoryinstitution of i.

FERM BP-10010

(4)

i. Name and address of depository institution to which the biologicalmaterial of interest is deposited

International Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology

Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan (post code:305-8566)

ii. Date when the biological material was deposited to the depositoryinstitution of i.

Feb. 24, 2004 (original deposit date)

iii. Accession number for the deposition assigned by the depositoryinstitution of i.

FERM BP-08642

1. A method of specifically determining a glycated β-chain N-terminal ofglycated hemoglobin, the method comprising: reacting the glycatedhemoglobin with enzymes comprising (i) a protease comprisingcarboxypeptidase B, neutral protease, carboxypeptidase A, thermolysin,Bacillus sp. ASP842 protease, Lysobacter enzymogenes YK-366 protease, orAeromonas hydrophila NBRC 3820 protease, and (ii) a ketoamine oxidasehaving at least 75% sequence homology with SEQ ID NO: 1, without anoperation for increasing the purity or concentration of glycatedhemoglobin or a substance derived from glycated hemoglobin; wherein theketoamine oxidase (ii) has a reactivity toε-1-deoxyfructosyl-(α-benzyloxycarbonyl-L-lysine) of 30% or lesscompared with that to 1-deoxyfructosyl-L-valyl-L-histidine.
 2. Thedetermination method according to claim 1, wherein the method isperformed under a reaction condition of pH 5.0 to 6.0.
 3. Thedetermination method according to claim 1, wherein the method isperformed under a reaction condition of pH 5.5 to 6.5.